Enrichments initiated with marine and estuarine sediments from the Arthur Kill (NY/NJ estuary) and Paleta Creek (San Diego Bay, CA), transformed 2,4-dichlorophenoxyacetic acid to 4-chlorophenol in the presence of sulfate (25 mM). Transformation of 2,4-dichlorophenoxyacetic acid was not observed with two other marine sediments from Tuckerton, NJ and Flax Pond (Stony Brook, NY). The lag period prior to 2,4-dichlorophenoxyacetic acid loss or the time required for 50% removal of 2,4-dichlorophenoxyacetic acid (t50) in Arthur Kill and San Diego enrichments was not affected by the presence of sulfate, although the addition of hydrogen and acetate decreased the lag periods and t50 values in these enrichments. The first step in the transformation of 2,4-dichlorophenoxyacetic acid was side-chain removal forming 2,4-dichlorophenol which was then dechlorinated to 4-chlorophenol. Second-generation Arthur Kill cultures dechlorinated 2,4-dichlorophenoxyacetic acid to 4-chlorophenoxyacetic acid. This dechlorination occurred both in the presence and absence of sulfate (25 mM); however, sulfate reduced the rate of dechlorination by approximately 50%. Second-generation cultures inoculated with the sulfate-amended enrichments consumed sulfate and dechlorinated 2,4-dichlorophenoxyacetic acid to 4-chlorophenoxyacetic acid concurrently when supplied with hydrogen and acetate; the rate of dechlorination was twice that of cultures that did not receive hydrogen and acetate. Sulfate consumption occurred only in cultures supplemented with hydrogen and acetate. These results indicate that dechlorination of an organochlorine pesticide in marine and estuarine sediments occurs in the presence of sulfate and the addition of readily utilizable carbon and electron donors can stimulate dechlorinating activity.
Various halogenated industrial chemicals, including organochlorine pesticides, have been shown to accumulate in marine and estuarine sediments [1–4]. The sources of these compounds include agricultural run-off and industrial and municipal waste discharge . Brominated, chlorinated and iodinated organic compounds found in marine and estuarine sediments are also produced by marine biota [6, 7]. The accumulation of halogenated pollutants and natural products in marine and estuarine sediments potentially selects for anaerobic bacteria which can transform these types of compounds. Under anaerobic conditions, halogenated aromatic compounds can undergo various transformations. For example, they can be dehalogenated or degraded via ring cleavage to CO2. Reductive dehalogenation reactions catalyzed by anaerobic bacteria are either co-metabolic processes or linked to respiration; a process termed dehalorespiration . In the process of dehalorespiration, an anaerobic bacterium utilizes a halogenated compound as a terminal electron acceptor, the reduction of which is coupled to ATP production.
A number of studies have demonstrated that marine or estuarine sediments can support reductive dehalogenation of halogenated phenols [9–16], benzoates [10, 14], catechols , and polychlorinated biphenyls [18–20]. In addition, studies show that these dehalogenation reactions occur in the presence of sulfate [9, 10, 13, 14], an abundant anion in marine systems . Sulfate, of course, can serve as an alternative terminal electron acceptor. In contrast, sulfate and sulfoxyanions have been shown to inhibit reductive dehalogenation reactions in enrichments initiated with inocula from non-marine and estuarine environments [22–28].
The negative impact of organochlorine pesticides on marine biota is well-documented [29, 30]. In contrast, the transformation of these compounds by anaerobic bacteria in marine and estuarine sediments has received little attention compared to other environments, such as groundwater aquifers, soils, pond and river sediments and sewage sludge (see  references there in). Our objective was to determine the factors that influence the transformation of a model organochlorine pesticide, 2,4-dichlorophenoxyacetic acid (2,4-D), in marine and estuarine sediments. We investigated the fate of 2,4-D in four different marine and estuarine sediments and examined the effects of sulfate, hydrogen and acetate on 2,4-D transformation.
2Materials and methods
The various sediments used as inocula in the present study are described in Table 1. The sediments were collected as cores (Arthur Kill, NY and San Diego, CA) or grab samples (Flax Pond, NY and Tuckerton, NJ) stored at 4°C in glass jars filled to capacity until used. These sediments were used to initiated enrichments with either 2,4-D alone (unamended), with 2,4-D and sulfate or with 2,4-D, hydrogen and acetate.
Table 1. Description of sediment samples used as inocula for enrichment cultures
Sediment ‘name’ and location
aPercent carbon and nitrogen were determined using a Carlo Erba NA1500 elemental analyzer.
‘Arthur Kill’ NY/NJ estuary near Fresh Kills landfill
Polyaromatic hydrocarbons, BTEX, pesticides, heavy metals, PCBs [3, 4]
Submerged surface (top 4 in.) marine sediment at Paleta Creek
‘Flax Pond’ Stony Brook, NY
Submerged mixed surface and subsurface marine sediment
‘Tuckerton’ Tuckerton, NJ
Subsurface marine sediment from intertidal zone collected at low tide
Enrichment cultures were prepared in anaerobic saline media . Strict anaerobic technique was used throughout. The medium was boiled for 15 min and cooled under a steady stream of N2:CO2 (70:30). Once cooled, sediment inocula (10% v/v) were added and 30 ml aliquots were dispensed into 60 ml serum bottles with N2:CO2 as the headspace and sealed with butyl rubber septa and aluminum crimps. Sets of triplicate bottles were supplemented with the following: 2,4-D only; 2,4-D and sulfate; 2,4-D, H2 and acetate; H2, acetate and sulfate only; sulfate only; or no supplements. Sterile controls were autoclaved on 3 consecutive days for 1 h and spiked with 2,4-D and sulfate. Sulfate and acetate were added from sterile stock solutions to final concentrations of 25 mM. 2,4-D (97%, Aldrich, Milwaukee, WI) was added from a sterile stock solution prepared in 0.1 N NaOH to a final aqueous concentration of 250 μM. Hydrogen was added by evacuating the culture headspace under vacuum and repressurizing the bottle to 14 psi with H2:CO2 (80:20). All enrichments were incubated in the dark at 30°C without shaking.
Bottles were well mixed and slurry samples (0.5–1.0 ml) were collected using sterile, degassed syringes, centrifuged for 2 min in a benchtop microfuge and the aqueous fraction filtered using a 0.45-μm Millex syringe filter (Millipore, Bedford, MA) prior to analysis. Removal of 2,4-D and production of metabolites was monitored using a high performance liquid chromatograph (Beckman, Fullerton, CA) equipped with a 4.6 mm×25 cm Ultrasphere C18 column (Beckman) with UV detection (283 nm). The initial mobile phase composition was 90% mobile phase A (40:57:3 methanol:water:acetic acid) and 10% mobile phase B (80:18:2 methanol:water:acetic acid) at a flow rate of 1.0 ml min−1 for 15 min then changed to 40% A:60% B over 12.5 min using a linear gradient and held for 2.5 min. The retention times (minutes), determined with standards, were as follows: phenol (9.0), 2-chlorophenoxyacetic acid (11.6), 2-chlorophenol (11.6), 4-chlorophenol (4-CP, 15.5), 4-chlorophenoxyacetic acid (4-CPA, 16.3), 2,4-D (26.9), 2,4-DCP (28.2). Sulfate concentrations were measured using an ion chromatograph as previously described .
Triplicate Arthur Kill enrichments (approximately 15 ml each) which received 2,4-D only, 2,4-D and sulfate, or 2,4-D, H2 and acetate were combined into one bottle for each type of enrichment using sterile, degassed 30-ml syringes after 144 days of incubation. These 45-ml pooled cultures were then distributed (10 ml each) to four sterile, 28-ml culture tubes crimp sealed with butyl rubber stoppers and degassed with N2:CO2, thus generating four second-generation cultures from each enrichment condition. These second-generation cultures were then supplemented as indicated in Table 2.
Table 2. Inocula and supplementation scheme for second-generation Arthur Kill cultures
Initial enrichment used as inoculuma
Additions to second generation culturesb
aInitial three replicates were pooled, then subdivided to four tubes.
bPairs of tubes received the indicated additions. Sulfate and acetate added to approximately 25 mM; 2,4-D added to 200 μM. H2:CO2 (80:20) at 14 psi, 13 ml headspace in 28-ml culture tube.
2,4-D and SO42−
2,4-D and sulfate
2,4-D, H2 and acetate
2,4-D and H2 and acetate
H2 and acetate
3.1Transformation of 2,4-D by anaerobic bacteria in marine and estuarine sediments
Enrichments with Arthur Kill and San Diego sediments, both with a history of exposure to contaminants (Table 1) initiated 2,4-D transformation within 80 days. Flax Pond and Tuckerton sediment enrichments did not exhibit any 2,4-D loss within 160 days (Fig. 1). Complete 2,4-D loss in Arthur Kill and San Diego sediment enrichments that received only 2,4-D occurred within 60 or 145 days, respectively (Fig. 1A). The presence of sulfate in the enrichments did not affect the rate or extent of 2,4-D transformation in these sediments (Fig. 1B). The addition of hydrogen and acetate, however, stimulated the transformation of 2,4-D in both the Arthur Kill and San Diego sediments with complete 2,4-D loss occurring within 70 days for both sediments (Fig. 1C). Neither the presence of 25 mM sulfate (Fig. 1B) nor hydrogen and acetate (Fig. 1C) stimulated any activity in enrichment cultures inoculated with sediments from Flax Pond and Tuckerton. No 2,4-D or sulfate loss was observed in sterile controls for all four sediments (data not shown).
The onset of 2,4-D transformation occurred much more rapidly in the Arthur Kill enrichments (38 days) than in the San Diego enrichments (77 days; Fig. 1A,B, Table 3). Likewise, the time required for 50% removal of 2,4-D in the Arthur Kill enrichments (49 days) was much shorter than for the San Diego enrichments (110 days; Table 3). Neither the lag periods nor the t50 values were affected by the addition of sulfate, indicating that sulfate had no effect on the establishment of microbial communities capable of transforming 2,4-D. The longer lag periods and t50 values for the San Diego sediments may be the result of lower concentrations of carbon and nitrogen as compared to the Arthur Kill sediments (Table 1).
Table 3. Summary of results from experiments with Arthur Kill and San Diego sediments
Initially observed intermediate(s)d
aSulfate and acetate added to approximately 25 mM; H2 added as H2:CO2 (80:20) at 14 psi, 20 ml headspace in 50-ml culture bottle. 2,4-D added to 200 μM.
bFirst time point where both 2,4-D loss (10%) and initial intermediate(s) formation (detection limit, 2 μM) were detected.
cTime, in days, for 2,4-D concentration to reach 50% of initial concentration.
The results from enrichments supplemented with hydrogen and acetate suggest that the differences observed between the Arthur Kill and San Diego unamended enrichments were a result of carbon limitation. The addition of hydrogen and acetate resulted in equivalent lag periods (10 and 14 days) and t50 values (32 and 30 days) for the Arthur Kill and San Diego enrichments, respectively (Table 3). Methane production was observed in these enrichments (Table 3). No detectable methane, however, was observed in the absence of hydrogen and acetate, thus 2,4-D transformation and dehalogenation (see below) in these sediments does not require active methanogenic populations.
The first transformation product observed in the Arthur Kill enrichments was 2,4-DCP (Table 3), suggesting that the initial step in the transformation of 2,4-D was side-chain removal. Subsequent dechlorination of 2,4-DCP resulted in the accumulation of 4-chlorophenol (Table 3). The initial intermediate observed in the San Diego enrichments was 4-chlorophenol with the exception of the sulfate-amended cultures in which both 2,4-DCP and 4-CP were detected (Table 3). This demonstrates that both dehalogenation and side-chain cleavage occurred, although the sequence of these reactions cannot be determined because an intermediate (i.e. 2,4-DCP or 4-CPA) was not detected. The initial detection of both 4-CP and 2,4-DCP in the sulfate-amended enrichments suggests that removal of the side chain preceded dechlorination. No loss of 4-CP was observed and phenol was not detected in any of the enrichment cultures with incubations up to 170 days.
Dehalogenation of 2,4-DCP occurred in all enrichments supplemented with sulfate (Table 3) however, sulfate consumption did not occur (data not shown). Control enrichments of all sediments supplemented with hydrogen, acetate, and sulfate, but without 2,4-D consumed sulfate, indicating that viable sulfate-reducing bacteria were present in all of the sediments. Sulfate consumption (>5 mM) did not occur in control enrichments supplemented with only sulfate (data not shown), suggesting that the sulfate-reducing bacteria were carbon limited and could not access the carbon and electron donors that were components of the sediments, or that the amount of carbon present in the sediments was insufficient to support >5 mM sulfate consumption. Reductive dechlorination, however, of approximately 200 μM 2,4-dichlorophenol to 4-chlorophenol occurred in the absence of any additional carbon or electron donor (Table 3), suggesting that there was sufficient utilizable carbon to support this amount of dehalogenation.
3.2Effect of sulfate on 2,4-D dechlorination
The Arthur Kill enrichment cultures were utilized for further experiments to confirm the effects of sulfate, hydrogen and acetate additions on 2,4-D transformation. The triplicate bottles for each enrichment culture, 2,4-D only, 2,4-D with sulfate and 2,4-D with hydrogen and acetate, were used to initiate second-generation cultures. These cultures were supplemented as indicated in Table 2.
Second-generation cultures inoculated with initial unamended enrichments were supplemented with 2,4-D or with 2,4-D and sulfate to further investigate the effect of sulfate on 2,4-D transformation. Dechlorination of 2,4-D occurred under both conditions, resulting in the transient accumulation of 4-CPA (Fig. 2A,B). The rate of dechlorination (4-CPA production) was approximately 50% slower in the sulfate-amended cultures, even though no sulfate consumption was observed (Fig. 2B). Also, complete removal of 2,4-D occurred within 40 days in the absence of sulfate and in 62 days in the presence of sulfate. As previously observed, 4-chorophenol was the final transformation product.
3.3Effect of hydrogen, acetate and sulfate on 2,4-D dechlorination
The effect of hydrogen and acetate addition on 2,4-D transformation in the presence of sulfate was tested using second-generation cultures inoculated with the sulfate-amended Arthur Kill enrichments. These second-generation cultures were supplemented with 2,4-D or with 2,4-D, hydrogen and acetate with results shown in Fig. 3. The sulfate added when the inoculating enrichments were prepared was still present at a concentration of approximately 25 mM. In the absence of hydrogen and acetate (Fig. 3A), 2,4-D was dehalogenated to 4-CPA with 4-CP as the final transformation product. Minor amounts of 2,4-DCP were produced. A slight decrease in sulfate concentration was observed (∼11%). The addition of hydrogen and acetate, however, resulted in concurrent sulfate consumption and dehalogenation (Fig. 3B). The rates of sulfate consumption and 2,4-D dehalogenation were approximately 1.8 mmol liter slurry−1 day−1 and 10 μmol liter slurry−1 day−1, respectively. In comparison, the rate of 2,4-D dehalogenation in the enrichments without hydrogen and acetate supplementation was approximately 5 μmol liter slurry−1 day−1.
3.4Effect of hydrogen and acetate on 4-chlorophenol transformation
Second-generation cultures were also initiated using Arthur Kill enrichments supplemented with hydrogen and acetate as the inoculum. These were used to determine if the accumulation of 4-CP in the initial enrichments was the result of carbon limitation. No 4-CP loss was detected in second-generation cultures in the presence or absence of hydrogen and acetate (data not shown).
Marine environments contain diverse halogenated compounds originating from either the metabolic activity of indigenous biota [6, 7] or from the actions of humans [1–4]. The abundance of organohalides suggests that long-term exposure to these compounds has provided a selective pressure for the evolution of dehalogenating mechanisms in anaerobic bacteria present in marine sediments. Evidence supporting this hypothesis includes the observation of bromo- and chlorophenol dehalogenating activity in marine sediment [9, 14] and the isolation of a dehalorespiring Desulfovibrio sp. from estuarine sediments . Our current findings offer further support and clearly demonstrate that anaerobic bacteria in marine and estuarine sediments can transform 2,4-D via reductive dechlorination and side-chain removal forming 4-CP (Fig. 4). Side-chain removal precedes dechlorination in the initial enrichments while it occurs after dechlorination in second-generation cultures (inoculated with enrichments exhibiting dechlorinating activity); this observation suggests that two different microbial populations are responsible for each reaction. These transformations were stimulated by the addition of exogenous carbon and electron donors and occurred in the presence of high levels of sulfate (25 mM), which is expected to be the predominant electron acceptor in marine environments [21, 34, 35]. Although transformation of 4-CP was not observed in this study, previous work [11, 12, 15] has demonstrated that 4-CP degradation to CO2 can be coupled to sulfate reduction in estuarine sediments. The lack of 4-CP dehalogenating activity may be a result of the long incubation period for the primary enrichment (144 days) prior to use in the second-generation cultures in addition to the use of 2,4-D, not 4-CP as the initial enrichment substrate. The lack of selective pressure over such a period of time may have resulted in the loss of the 4-CP dehalogenating population. Alternatively, the population may have never existed in the original sediment sample.
The presence of sulfate had no effect on the onset or rate of 2,4-D transformation in our enrichments with marine and estuarine sediments. Cultures prepared with or without sulfate exhibited similar lag periods and t50 values (Fig. 1B, Table 3). In contrast, it has been reported that 2,4,5-T dechlorinating activity in enrichments using aquifer material from methanogenic or sulfidogenic areas within a landfill site were completely inhibited by the addition of 29 mM sulfate . It was further reported that supplementation with various carbon donors shortened acclimation times in similar enrichments with or without sulfate. Acclimation times, however, were consistently longer in the presence of sulfate . Indeed, marine and non-marine dehalogenating communities may respond differently to the presence of sulfate. In this present study, second-generation Arthur Kill cultures dehalogenated 2,4-D to 4-CPA within 40 days of incubation in the presence (Fig. 3A) and absence (Fig. 2A) of sulfate. We did, however, observe a decrease in the rate of 2,4-D dechlorination to 4-CPA when sulfate was added to second-generation cultures inoculated with enrichments that did not receive sulfate initially. Similarly, utilizing an acclimated, methanogenic, freshwater pond sediment enrichment, Kohring et al. , observed that dechlorination rates of 2,4-DCP (to 4-CP) in the presence of 25 mM sulfate were slower than in the absence of sulfate.
In the results presented here, simultaneous sulfate consumption and dechlorination were only observed in the presence of exogenous carbon and electron donors. Kohring et al.  observed concurrent sulfate reduction and dehalogenation without exogenous carbon donor in freshwater pond sediment enrichments, suggesting that higher concentrations of utilizable carbon donors were present in those sediments. Gibson and Suflita  observed that the addition of acetate to enrichments using sulfidogenic aquifer material allowed for dechlorination of 2,4,5-T, leading to the hypothesis that the dechlorinating bacteria could not compete successfully with sulfate-reducing bacteria for reducing equivalents provided by the aquifer material. In contrast, we observed dechlorination (and side-chain cleavage), but not sulfate reduction, in enrichments that were not supplied hydrogen and acetate, suggesting that the dechlorinating bacteria in these sediments can successfully compete with the sulfate-reducing bacteria for reducing equivalents.
An alternative hypothesis is that the observed reductions are catalyzed by the same bacterium and that under carbon and electron limiting conditions, dehalogenation reactions capture the metabolic pool of reducing equivalents. A number of dehalorespiring bacteria are capable of using sulfate or other sulfoxyanions as electron acceptors (, for review see ). The observation that both sulfate reduction and dehalogenation occur in the presence of excess carbon and electron donors does not exclude either the intracellular or interspecies competition for reducing equivalents.
There were distinct differences between the two active sediments with respect to the lag period prior to 2,4-D loss and the time required for 50% removal. The Arthur Kill enrichments had lower lag and t50 values compared to San Diego enrichments. These differences could have been caused: (1) by higher concentrations of readily utilizable carbon and electron donors present in the Arthur Kill sediments; (2) by different bacterial communities, with inherently different rates of 2,4-D transformation; or (3) by differences in the initial size of the populations capable of transforming 2,4-D. We observed that the lag and t50 values were substantially decreased to equivalent values when hydrogen and acetate were supplied. Also, shorter lags and lower t50 values were obtained with the Arthur Kill sediments which had 4.4 times higher organic carbon than the San Diego sediments, suggesting that readily utilizable carbon and electron donors were limiting in the San Diego sediments.
Our findings document that the organochlorine pesticide 2,4-D can be reductively dechlorinated in marine and estuarine sediments in the presence of sulfate. These observations add to the mounting evidence that not all reductive dehalogenation reactions are inhibited by sulfate. The demonstration of concurrent reductive dehalogenation and sulfate reduction suggests that reductive dehalogenation can perhaps be considered a viable intrinsic remediation strategy in marine and estuarine systems despite the high concentrations of sulfate. Interestingly, the two sediments without any prior history of contamination, Tuckerton and Flax Pond, exhibited no 2,4-D transformation activity even with hydrogen and acetate addition. Further studies with different marine and estuarine sediments, both contaminated and prisitine, and other organochlorine pesticides can help determine if the transformation reactions we observed are limited to contaminated sediments and if structurally analogous pesticides can undergo similar transformations.
We would like to thank Monica Togna of Rutgers University, Beau Ranheim and the crew of the Osprey of the New York City Department of Environmental Protection and Sabine E. Apitz and Victoria J. Kirtay of the Space and Naval Warfare Systems Center-San Diego for assistance in sediment collection. We would also like to thank Vivian Chu and Roza Wojcik for technical assistance and Gary Taghon for sediment analyses. This research was funded in part by awards R-823575 from the United States Department of Environmental Protection and N00014-94-1-0434 from the Office of Naval Research.