Microbial Dechlorination Alleviates Inhibitory Effects of PCBs on

Toxicities of the parent Aroclor mixtures and their dechlorinated products were evaluated using in vitro fertilization of mouse gametes, as PCBs have ...
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Research Communications Microbial Dechlorination Alleviates Inhibitory Effects of PCBs on Mouse Gamete Fertilization in Vitro M A H M O U D A . M O U S A , †,‡ J O H N F . Q U E N S E N I I I , †,‡ K A R E N C H O U , ‡,§ A N D S T E P H E N A . B O Y D * ,†,‡ Department of Crop and Soil Sciences, Department of Animal Science, and Institute for Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824

Introduction It is estimated that approximately 1.4 billion lb of polychlorinated biphenyls (PCBs) have been produced worldwide and that several hundred million pounds have been released into the environment (1). Commercial PCBs were manufactured and used as complex mixtures that typically consisted of 60-90 (of a possible 209) PCB congeners differing in the position and number of chlorines on the biphenyl structure (2). Although the use of PCBs has been highly restricted since the 1970s, PCBs are ubiquitous environmental contaminants (3). Because of widespread occurrence of PCBs in the environment and their tendency to accumulate in biological tissues, much research on the toxicity of commercial PCB mixtures has been performed. PCBs are known to elicit a spectrum of toxic responses in humans and laboratory animals including lethality, reproductive and developmental toxicity, porphyria, body weight loss, dermal toxicity, immunosuppressive effects, hepatotoxicity, neurotoxicity, thymic atrophy, carcinogenesis, and disruption of the homeostasis of steroid hormones (4). Reproductive failures linked to PCBs have been observed in several mammalian species (5). Embryonic abnormality and low fertility occurred after gestational and neonatal exposure of rodents to PCBs (6, 7). Increases in the uterine weight and uterine glycogen were observed in female rats exposed to commercial PCBs (8, 9). Increased testicular weight was observed in rats exposed to Aroclor 1254 before weaning and in mice exposed to 2,2′,4,4′,5,5′-hexaCB during gestation (10, 11). In vitro, PCBs have been shown to directly inhibit the fertilization of mouse gametes (12). PCBs are generally considered as persistent environmental contaminants, primarily because of the inability of native microbial populations to degrade many PCB congeners in aerobic habitats. More recently, the reductive dechlorination of PCBs by bacteria found in anaerobic * Corresponding author telephone: (517) 355-3993; fax: (517) 3550270. † Department of Crop and Soil Sciences. ‡ Institute for Environmental Toxicology. § Department of Animal Science.

S0013-936X(95)00929-1 CCC: $12.00

 1996 American Chemical Society

habitats (e.g., sediments) has been recognized as an important environmental fate (13-20). Reductive dechlorination of PCBs involves removal of chlorine directly from the biphenyl ring and replacement with hydrogen. This process results in a dechlorinated mixture in which the average number of chlorines per biphenyl is substantially reduced. Generally, chlorines in the meta and para positions are most readily removed. In situ anaerobic reductive dechlorination of PCBs has now been documented at numerous sites including the Hudson River (NY), Silver Lake (MA), Sheboygan River (WI), Waukegan Harbor (IL), Acushnet Estuary (MA), Hoosic River (MA), and River Raisin (MI) (14). Anaerobic reductive dechlorination of commercial Aroclors may substantially alter the toxic effects of these mixtures, potentially causing an increase, decrease, or shift in the spectrum of toxicity. This, coupled with the increasing use of toxicity-based risk assessment of contaminated sites (21), emphasizes the need to evaluate the toxicity of microbially dechlorinated PCB mixtures. This is critical to an accurate assessment of the risks associated with PCBs that have undergone reductive dechlorination via native microbial populations (e.g., Upper Hudson River) and what remedial action should be taken. In this study, Aroclor 1242 and Aroclor 1254 were dechlorinated by microorganisms eluted from PCBcontaminated Silver Lake (SL) and River Raisin (RR) sediments. Toxicities of the parent Aroclor mixtures and their dechlorinated products were evaluated using in vitro fertilization of mouse gametes, as PCBs have previously been demonstrated to inhibit this process (12).

Methods PCB Dechlorination. Aroclor 1242 and Aroclor 1254 were dechlorinated by microorganisms extracted with reduced anaerobic mineral medium (RAMM) from contaminated sediments of RR and SL (20, 22). Dechlorination was carried out under anaerobic conditions in sealed 100-mL vials containing non-PCB-contaminated sediments (75 g) from the Red Cedar River (Okemos, MI). These sediments were amended with PCBs (600 µg/g of sediment) and inoculated with the RR or SL microorganisms. In addition to the actively dechlorinating microorganisms, a control containing autoclaved microorganisms together with PCBs and a control containing active microorganisms with no PCBs were included. The incubation was continued anaerobically for a period of 9 months. The contents of the 100-mL vials were transferred into 1000-mL vials, and then PCBs were recovered by extraction twice with 500 mL of acetone and three times with 500 mL of hexane-acetone (9:1). The extraction solvents were evaporated under N2, and the residues were dissolved in 100 mL of hexane. The hexane solution was washed with concentrated sulfuric acid (three times × 25 mL) followed by 2% sodium chloride solution (two times × 50 mL) then passed over a 50-mL Florisil copper column contained in a 100-mL buret (20). The total molar PCB concentration and the identity and concentra-

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TABLE 1

Concentrations (Total µmol/L) of PCB Congeners in Each Chromatographic Peak at the High Dose of Each PCB Test Mixturea Aroclor 1242

Aroclor 1254

peak

structure

auto

RR live

SL live

auto

RR live

SL live

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71

2342,2′-/2,62,4-/2,52,3′2,4′-/2,32,2′,63,4-/3,4′2,2′,5-/4,4′2,2′,42,3′,6-/2,3,62,2′,3-/2,4′,62′,3,5,-/(2,2′,6,6′-) 2,4,52,3′,52,3′,42,4′,52,4,4′-/(2,2′,4,6-) 2′,3,4-/2,3,3′-/2,2′,5,6′-/(2,3,4-) 2,3,4′-/(2,2′,4,6′-) 2,2′,3,62,2′,3,6′2,2′,5,5′-/(2,3′,5′,6-) 2,2′,4,5′2,2′,4,4′2,2′,4,5-/2,4,4′,63,3′,42,2′,3,5′2,2′,3,4′-/2,3,3′,6-/3,4,4′2,2′,3,4-/2,3,4′,6-/(2,3′,4′,6-/2,3′,5,5′-) 2,2′,3,6,6′2,2′,3,3′2,3′,4,5-/2,2′,4,4′,6-/(2,3,3′,5-/2,2′,4,5′,6-) 2,3,4′,5-/(2,3,3′,5′-) 2,4,4′,5-/(2,2′,3,5,6′-) 2,3′,4′,5-/(2′,3,4,5-) 2,2′,3,5′,6-/2,3′,4,4′-/(2,2′,4,5,6′-) 2,2′,3,4′,6-/(2,3,3′,4-) 2,2′,4,4′,6,6′-(surrogate) 2,3,3′,4′-/2,3,4,4′2,2′,4,5,5′-/2,2′,3,4′,52,2′,4,4′,52,3′,4,4′,6-/(2,2′,3,4′,6,6′-/2,3,3′,5,6-) 2,2′,3′,4,5-/(2,2′,3,4,5-/2,2′,3,5,6,6′-) 2,2′,3,4,5′-/2,3,4,4′,6-/(2,3,3′,5,5′-) 2,2′,3,4,4′2,2′,3,3′,6,6′3,3′,4,4′-/2,3,3′,4′,62,2′,3,5,5′,62,2′,3,3′,5,6′-/(2′,3,4,5,5′-/2,2′,3,4,5′,6-) 2,3′,4,4′,5-/2,2′,3,4′,5′,6-/2′,3,4,4′,52,2′,3,3′,5,6-/2,2′,3,3′,4,6-/2′,3,3′,4,52,2′,3,4′,5,5′-/(2,3,3′,4,5′,6-) 2,2′,4,4,′,5,5′2,2′,3,3′,4,6′-/2,3,3′,4,4′2,2′,3,4,5,5′2,2′,3,3′,5,6,6′2,2′,3,3′4,5′2,2′,3,3′,4,6,6′-/2,2′,3,4,4′,52,2′,3,4,4′,5′-/2,3,3′,4,5,6-/(2,3,3′,4′,5,6-) 2,3,3′,4,4′,62,2′,3,3′,5,5′,6-/2,2′,3,3′,4,52,2′,3,3′,4,5′,62,2′,3,4′,5,5′,6-/(2,2′,3,4,4′,5,6′-) 2,2′,3,4,4′,5′,62,2′,3,3′,4,4′-/2,3′,4,4′,5,5′2,2′,3,4,5,5′,62,2′,3,3′,4,5,6′-/(2,2′,3,4,4′,5,6-) 2,2′,3,3′,4′,5,62,2′,3,3′,4,4′,6-/2,3,3′,4,4′,5-/2,2′,3,3′,5,5′,6,6′-

0.59 0.00 0.00 3.29 0.79 1.13 4.85 0.50 0.31 6.16 3.08 0.71 3.30 0.08 0.16 0.94 0.79 4.46 5.84 4.56 2.35 0.89 0.40 2.50 1.97 0.91 1.09 0.07 2.24 2.65 2.46 0.01 0.73 0.18 0.05 1.08 2.95 2.89 0.36 0.00 4.49 1.07 0.32 0.00 0.31 0.38 0.17 0.01 0.92 0.37 0.07 0.62 0.01 0.02 0.07 0.22 0.00 0.01 0.01 0.01 0.13 0.02 0.01 0.00 0.01 0.00 0.01 0.00 0.01 0.00 0.01

6.28 0.00 2.91 13.83 1.48 3.16 20.08 2.51 0.54 0.57 12.53 1.21 3.85 0.42 0.04 0.26 1.08 0.38 2.05 0.32 0.26 0.00 0.10 0.26 0.31 1.64 0.00 0.00 0.04 0.10 0.03 0.00 0.02 0.01 0.01 0.01 0.02 0.04 0.00 0.00 0.05 0.04 0.01 0.00 0.00 0.01 0.00 0.00 0.02 0.01 0.00 0.03 0.00 0.00 0.01 0.01 0.00 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

4.71 0.00 0.55 12.46 1.30 3.93 10.65 2.14 1.33 1.84 6.42 1.68 3.41 0.48 0.02 2.52 4.18 5.59 6.83 0.41 0.80 0.02 0.20 1.21 1.32 1.21 0.00 0.00 0.23 0.38 0.06 0.00 0.07 0.01 0.05 0.02 0.03 0.08 0.02 0.00 0.14 0.10 0.01 0.00 0.00 0.02 0.00 0.00 0.05 0.01 0.00 0.02 0.00 0.00 0.02 0.01 0.00 0.04 0.00 0.00 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.22 0.02 0.04 0.19 0.02 0.01 0.14 0.10 0.01 0.07 0.00 0.00 0.02 0.03 0.19 0.14 0.11 0.03 0.04 0.02 2.71 0.62 0.16 0.05 0.00 0.99 0.18 0.51 0.01 0.11 0.01 0.01 0.37 1.97 2.69 1.45 0.00 1.73 8.38 2.15 0.02 2.06 2.76 0.63 0.46 7.76 1.70 1.03 1.73 0.32 0.58 2.91 1.99 0.60 0.14 0.43 0.20 6.16 0.77 0.31 0.00 0.25 0.16 0.62 0.02 0.25 0.15 0.60

0.32 0.00 0.65 5.01 0.26 0.19 1.97 3.47 0.05 0.26 15.06 0.41 5.70 0.79 0.49 0.03 0.23 0.00 1.67 1.74 0.00 0.00 0.07 0.62 0.91 13.19 0.00 0.03 0.04 0.11 0.29 0.00 0.12 0.41 0.02 0.05 0.14 0.44 0.10 0.00 0.21 0.61 0.23 0.00 0.10 0.13 0.04 0.06 0.44 0.20 0.17 0.32 0.05 0.13 0.70 0.21 0.12 0.03 0.08 0.04 1.36 0.16 0.07 0.01 0.07 0.05 0.10 0.01 0.08 0.05 0.16

0.61 0.00 0.00 4.00 0.22 0.26 0.40 2.63 0.00 0.75 5.92 3.39 2.49 0.24 0.24 0.63 2.74 0.98 3.29 1.00 0.00 0.01 0.55 2.25 4.49 10.93 0.00 0.02 0.11 0.39 0.24 0.13 0.18 0.33 0.14 0.14 0.23 0.78 0.38 0.00 0.84 1.56 0.33 0.00 0.17 0.29 0.06 0.12 0.83 0.36 0.24 0.46 0.12 0.17 0.85 0.29 0.21 0.00 0.10 0.05 1.72 0.19 0.08 0.01 0.08 0.06 0.15 0.01 0.09 0.05 0.19

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Table 1 (Continued) Aroclor 1242

Aroclor 1254

peak

structure

auto

RR live

SL live

auto

RR live

SL live

72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87

2,2′,3,3′,4,5,6-/2,3,3′,4,4′,5′-/2,2′,3,3′,4,5′,6,6′2,2′,3,3′,4,5,5′-/(2,3,3′,4,5,5′,6-) 2,2′,3,4,4′,5,5′2,3,3′,4′,5,5′,62,3,3′,4,4′,5′,62,2′,3,3′,4,5,6,6′2,2′,3,3′,4,4′,5-/2,3,3′,4,4′,5,62,2′,3,3′,4,5,5′,62,2′,3,3′,4′,5,5′,62,2′,3,3′,4,4′,5,6′-/2,2′,3,4,4′,5,5′,62,3,3′,4,4′,5,5′2,2′,3,3′,4,4′,5,62,2′,3,3′,4,5,5′,6,6′2,2′,3,3′,4,4′,5,5′2,3,3′,4,4′,5,5′,62,2′,3,3′,4,4′,5,5′,6total

0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 76.63

0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 76.63

0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 76.63

0.08 0.07 0.48 0.02 0.02 0.00 0.25 0.01 0.00 0.03 0.01 0.01 0.00 0.02 0.00 0.01 61.16

0.02 0.02 0.16 0.01 0.00 0.00 0.08 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.01 61.16

0.03 0.03 0.18 0.01 0.00 0.00 0.10 0.00 0.00 0.01 0.00 0.01 0.00 0.01 0.00 0.01 61.16

a Parentheses around a structure indicate that the congener is not detectable in Aroclors. Control, non-dechlorinated; RR, dechlorinated by River Raisin microorganisms; SL, dechlorinated by Silver Lake microorganisms.

tion of the individual congeners were determined by GC (20). The congener composition was expressed as molar percentage of the total moles in each sample. The molar concentrations of the PCB congeners present in each chromatographic peak are given in Table 1. The different extracts were tested for toxicity to mouse gamete fertilization in vitro. Toxicity Evaluation. The success of in vitro fertilization of mouse gametes was measured in the presence of 10 and 20 ppm of non-dechlorinated (Aroclors 1242 and 1254) and their molar equivalent of dechlorinated PCBs. The designation “ppm” used for the dechlorinated PCBs, therefore, reflects the total molar concentration of the starting material and not the actual weight of the dechlorinated products. The total micromolar dosages (micromolar concentration in the culture medium) were 76.63 and 38.32 for Aroclor 1242 and 61.16 and 30.58 for Aroclor 1254. The test materials were added to the culture medium, BMOC-3 (23), for both gamete collection and insemination. Despite being added in excess of their water solubilities, PCBs remained in the aqueous phase due to their association with serum components (24). Sperm were collected from epididymides of B6D2-F1 male mice (Jackson Laboratory, Bar Harbor, ME). This crossbreed, from C57BL/6J females and DBA/2J males, has been shown to be responsive to dioxin and Arochlor 1254 in CYP1A induction (25). Each pair of epididymides was excised and submersed in 1 mL of the test medium containing the test material. Sperm were released by poking the epididymides with a 25-G precision glide needle and incubating for 1 h at 37 °C and 5% CO2. Tissue debris was removed after 30 min to minimize the partitioning of the test compounds into the tissue. Eggs were obtained from 5-7-week-old B6D2-F1 female mice superovulated with intraperitoneal injections of 10 IU of pregnant mare’s serum gonadotropin (PMSG) followed 4850 h later by 10 IU of human chorionic gonadotropin (hCG). Thirteen hours after hCG administration, cumulus-enclosed eggs were collected in 1 mL of BMOC-3 also containing the test material. The eggs were then inseminated with 50 µL of sperm suspension. The final sperm concentrations were 0.5 to 1.5 × 106 cells/mL. After 25-27 h incubation, 50 µL of 35 µM bisbenzimide trihydrochloride (Sigma Chemical Co, St. Louis, MO) was added to the gametes for nuclear

staining. After further incubation for 30 min, the eggs were washed in 0.5 mL of BMOC-3 without bovine serum albumin and assessed for the success of fertilization by an invertedphase contrast microscope (200×). The egg fertilization and degeneration were inspected by inverted-phase contrast microscopy. Eggs at the two-cell stage or the one-cell stage with two pronuclei and a second polar body were considered fertilized. Fragmented, degenerated, and onecell eggs without multiple nuclei were considered nonfertilized. The results of the in vitro fertilization were analyzed by the χ2 square procedure (MINITAB Inc, State College, PA) at the 0.05 significance level.

Results and Discussion The congener profiles of the original Aroclors and the dechlorinated products are presented in Figure 1 as histograms showing the molar percentage of PCB congeners associated with each chromatographic peak. In general, anaerobic incubation decreased the molar percentage of the more chlorinated congeners and increased that of the less chlorinated congeners. Dechlorination of Aroclor 1242 by the RR organisms removed an average of 1.06 chlorine atoms per biphenyl, which corresponds to 56% of the meta and para chlorines. There was no evidence of ortho chlorine removal. The predominant dechlorination products (i.e., those increasing to more than 5 mol %) contained chlorine at ortho and para positions, including 2-CB, 2,2′-diCB/2,6-diCB, 2,4′diCB/2,3-diCB, and 2,2′,4-triCB (Figure 1). These congeners comprised 15.5 mol % of the parent Aroclor, but were 68.8 mol % of the dechlorination products. Less prominent products (i.e., those increasing to less than 5 mol %) were 4-CB, 2,4-diCB/2,5-diCB, 2,3′-diCB, 2,2′,6-triCB, 3,4-diCB/ 3,4′-diCB, 2,3′,6-triCB/2,3,6-triCB, 2,2′,3-triCB/2,4′,6-triCB, 2′,3,5-triCB, 2,3′,4-triCB, and 2,2′,4,4′-tetraCB, which comprised 11.1 mol % of the parent Aroclor and 24.5 mol % of the dechlorination products. Such dechlorination pattern has been previously described as process M and is characterized by the removal of flanked meta chlorines (those which have adjacent positions occupied by chlorine) as well as nonflanked meta chlorines (14). Thus, it is probable that most of the dechlorination by the RR microorganisms occurred from the meta positions.

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FIGURE 1. Mole percentages of PCB congeners in non-dechlorinated and dechlorinated PCB mixtures as represented by each chromatographic peak after about a 9-month incubation of Aroclor 1242 or Aroclor 1254 with autoclaved and live microorganisms eluted from the River Raisin (RR) or Silver Lake (SL). The peak numbering corresponds to the order in which the congeners were eluted; lower numbered peaks generally represent less chlorinated congeners. The dechlorinating microorganisms were mixed with non-PCB-contaminated Red Cedar sediment suspended in reduced anaerobic mineral medium (RAMM) (22). Aroclor 1242 or 1254 was added to sediment slurries containing either autoclaved or live inocula and incubated for about 9 months after which time PCBs were extracted, purified, and quantified. The PCB congeners eluting in each chromatographic peak are given in Table 1.

Dechlorination of Aroclor 1242 by SL microorganisms was less extensive than by RR microorganisms, with an average of 0.75 chlorine atoms per biphenyl or 40% of the meta and para chlorines being removed. The predominant dechlorination products were 2-CB, 2,2′-diCB/2,6-diCB, and 2,4′-diCB/2,3-diCB (Figure 1). These congeners totaled only 11.4 mol % of the parent Aroclor but were 36.3 mol % of the dechlorination products. The less prominent products included 4-CB, 2,4-diCB/2,5-diCB, 2,3′-diCB, 2,2′,6-triCB, 3,4-diCB/3,4′-diCB, 2,2′,4-triCB, 2,3′,6-triCB/2,3,6′-triCB, 2,2′,3-triCB/2,4′,6-triCB, 2′,3,5-triCB/2,2′,6,6′-tetraCB, 2,3′,5triCB, 2,3′,4-triCB, 2,4′,5-triCB, and 2,4,4′-triCB, comprising 29.8 mol % of the parent Aroclor and 54.2 mol % of the dechlorination products. SL microorganisms formed more 2,3′,5-triCB, 2,3′,4-triCB, and 2,4′,5-triCB than did RR microorganisms. This demonstrated that SL microorganisms were less able to remove nonflanked meta chlorines than were RR microorganisms. Microorganisms from the RR removed an average of 1.73 chlorine atoms per biphenyl (54% of the meta and para chlorines) from the congeners present in Aroclor 1254; again there was no evidence that any ortho chlorines were removed. The predominant products formed were 2,2′diCB/2,6-diCB, 2,2′,6-triCB, 2,2′,4-triCB, 2,2′,3-triCB/2,4′,6triCB, and 2,2′,4,4′-tetraCB (Figure 1). These congeners totalled only 1.0 mol % of the parent Aroclor but were 69.3 mol % of the dechlorination products. Less prominent products were 2-CB, 4-CB, 2,4-diCB/2,5-diCB, 2,3′-diCB, 2,4′-diCB/2,3-diCB, 3,4-diCB/3,4′-diCB, 2,2′,5-triCB/4,4′diCB, 2,3′,6-triCB/2,3,6-triCB, 2′,3,5-triCB/2,2′,6,6′-tetraCB,

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2,4,5-triCB, 2,3′,5-triCB, 2,3′,4-triCB, 2,4,4′-triCB/2,2′,4,6tetraCB, 2′,3,4-triCB/2,3,3′-triCB/2,2′5,6′-tetraCB/2,3,4triCB, 2,2′,3,6′-tetraCB, 2,2′4,5′-tetraCB, 3,3′,4-triCB, 2,2′,3,3′tetraCB, 2,3′,4,5-tetraCB/2,2′,4,4′,6-pentaCB/2,3,3′,5-tetraCB/ 2,2′,4,5′,6′-pentaCB, and 2,3,4′,5′-tetraCB/2,3,3′,5′-tetraCB comprising 2.4 mol % of the parent Aroclor and 17.4 mol % of the dechlorination products. Chlorine substitutions on these products were mainly at the ortho and para positions, which indicated that, as with Aroclor 1242, meta chlorines were removed from both flanked and nonflanked meta positions. Dechlorination of Aroclor 1254 by SL microorganisms was less extensive than that by RR microorganisms, with an average of 1.41 chlorines per biphenyl (44% of the meta and para chlorines) being removed. The predominant products formed included 2,2′-diCB/2,6-diCB, 2,2′,4-triCB, 2,3′,6-triCB/2,3,6-triCB, 2,4,4′-triCB/2,2′,4,6-tetraCB, 2,2′,4,5′-tetraCB, and 2,2′,4,4′-tetraCB. These congeners accounted for only 2.1 mol % of the parent Aroclor but 52.3 mol % of the dechlorination products. Less prominent products were 2-CB, 2,4-diCB/2,5-diCB, 2,3′-diCB, 2,4′diCB/2,3-diCB, 2,2′,6-triCB, 2,2′,5-triCB/4,4′-diCB, 2,2′,3triCB/2,4′,6-triCB, 2′,3,5-triCB/2,2′,6,6′-tetraCB, 2,4,5-triCB, 2,3′,5-triCB, 2,3′,4-triCB, 2,4′,5-triCB, 2′,3,4-triCB/2,3,3′triCB/2,2′,5,6′-tetraCB/2,3,4-triCB, 2,2′,3,6′-tetraCB, 3,3′,4triCB, 2,2′,3,4-tetraCB/2,3,3′,6-tetraCB/3,4,4′-triCB, 2,2′,3,3′tetraCB, 2,3′,4,5-tetraCB/2,2′,4,4′,6-pentaCB/2,3,3′,5-tetraCB/ 2,2′,4,5′,6-pentaCB, and 2,3,4′,5-tetraCB/2,3,3′,5′-tetraCB, which represented 1.9 mol % of the parent Aroclor and 24.5 mol % of the dechlorination products. Congeners con-

FIGURE 2. Fertilization and egg degeneration of mouse gametes in the presence of parent Aroclor 1242 (incubated with and then extracted from autoclaved sediment slurries) and of Aroclor 1242 dechlorinated by either River Raisin (RR/1242) or Silver Lake (SL/ 1242) microorganisms. The parent Aroclors were tested at two dose levels (10 and 20 ppm), and the dechlorinated Aroclors were tested as total molar equivalents of these doses. PCBs were added to the culture medium (Brinster’s medium, BMOC-3) (22) for both gamete collection and insemination. The control treatments included an Aroclor positive control and three negative controls. The negative controls were extracts of active sediments containing no Aroclor, tert-butanol, and BMOC-3 without any additions. Only the BMOC-3 control is shown in the figure as the negative control; the results of active sediment and tert-butanol treatments were not significantly different from this control. The results of the Aroclor positive control (non-incubated and non-extracted) was not significantly different from that of the non-dechlorinated (autoclaved inoculum) Aroclor.

taining nonflanked meta chlorines made up a greater proportion of the dechlorination products of SL microorganisms than RR microorganisms. SL microorganisms produced more 2,3′,6-triCB/2,3,6-triCB, 2,3′,4-triCB, and 2,2′,4,5′-tetraCB than RR microorganisms. These differences again indicated that SL microorganisms were less capable of removing nonflanked meta chlorines than were RR microorganisms. In vitro fertilization assay of mouse gametes was used to assess the relative toxicities of the parent Aroclors and their dechlorinated products. The gametes were exposed to 0, 10, and 20 ppm of Aroclor 1242 or Aroclor 1254 and to equal total molar concentrations of the RR and SL dechlorinated products of each Aroclor. Exposure of gametes to the non-dechlorinated Aroclors resulted in two adverse effects: a high percentage of degenerated eggs and a low percentage of fertilized eggs. In the absence of PCBs, few eggs (1-7%) degenerated and most eggs (83-86%) were fertilized (Figures 2 and 3). Dechlorination of Aroclor 1242 markedly reduced or eliminated the adverse effects on mouse gametes (Figure 2). At the high dose (20 ppm), the parent Aroclor 1242 caused all eggs to degenerate and completely eliminated fertilization. However, in the presence of equivalent molar concentrations of the dechlorinated products, egg degeneration was essentially zero, equal to that in the controls without PCBs. Furthermore, the percent of fertilized eggs increased from zero to 63% ( 10 and 21% ( 5 in eggs exposed to RR and SL dechlorinated products, respectively.

FIGURE 3. Fertilization and egg degeneration of mouse gametes in the presence of non-dechlorinated Aroclor 1254 and Aroclor 1254 dechlorinated by either River Raisin (RR/1254) or Silver Lake (SL/ 1254) microorganisms. Treatments are similar to those of Aroclor 1242 as described in Figure 2.

At 10 ppm of the parent Aroclor 1242, few eggs degenerated, but only 35% ( 14 were fertilized. Dechlorination by RR microorganisms resulted in a fertilization rate that was similar to that of the control without PCBs, indicating complete alleviation of the toxic effects on fertilization at this concentration. In the presence of 10 ppm SL dechlorination products, the proportion of eggs fertilized did not differ significantly from that of Aroclor 1242. Dechlorination of Aroclor 1254 resulted in a similar reduction in the toxic effects of PCBs on mouse gametes (Figure 3). At 20 ppm of the parent Aroclor 1254, all eggs degenerated and hence no fertilization occurred. However, when exposed to the RR and SL dechlorination products at the equivalent dose, only 21% ( 12 and 16% ( 5 of the eggs degenerated, and 8% ( 2 and 31% ( 4 of the eggs were fertilized, respectively. Exposure to 10 ppm of the parent Aroclor 1254 also resulted in considerable egg degeneration (66% ( 21) and low egg fertilization (21% ( 17). At 10 ppm, dechlorination of Aroclor 1254 by RR microorganisms, as with Aroclor 1242, eliminated all observed toxic effects. Dechlorination of Aroclor 1254 by SL microorganisms achieved a similar effect, resulting in fewer degenerated eggs and an increase in the percentage of fertilized eggs. Enormous efforts have been made to correlate the health effects of PCBs with the number and position of chlorine substitutions on the biphenyl molecule (4). Generally, highly chlorinated congeners are more carcinogenic and immunotoxic than less chlorinated congeners (26). The coplanar congeners that induce dioxin-like activity are known for their hepatotoxicity (4, 26). Lesser chlorinated congeners such as 2,2′-diCB have been reported to be neurotoxic (27). Until now, attempts to evaluate the toxicity of microbial PCB dechlorination products were virtually nonexistent despite the obvious potential for dechlorination to alter the toxicity of PCB mixtures in contaminated sediments and soils and, hence, to impact the assessment of risk posed by such sites. Earlier we reported a decrease in the dioxin-like toxicity of commercial Aroclors due to microbial removal of meta and para chlorines (28). The

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dioxin-like toxicity, expressed as toxicity equivalents (TEQs), was based on the EROD (ethoxyresrufinO-dethylase) induction potencies of individual PCB congeners relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin. TEQs were calculated from measured concentrations of specific PCB congeners present in the original Aroclor and in the dechlorinated product mixtures (which were similar to those described here) and their EROD induction potencies. TEQs of commercial Aroclors determined in this fashion decreased by 64-97% due to the dechlorination by SL microorganisms. The current study demonstrates that the inhibitory effects of PCB mixtures on the fertilization of mouse gametes are strongly influenced by the specific congener composition of the mixture. Microbial reductive dechlorination of commercial Aroclors in anaerobic sediment slurries significantly lowered or completely alleviated the toxicities of these PCB mixtures. This was convincingly demonstrated by the high fertilization of mouse gametes in the presence of dechlorinated PCBs as compared to the parent PCB mixtures, which caused complete or substantial reduction in fertilization success. Generally, Aroclor 1254 and its dechlorination products were more toxic than Aroclor 1242 and its dechlorination products. For both Aroclors at the concentration of 10 ppm, observed toxicity was eliminated as a result of dechlorination by the RR microorganisms. For both the RR and SL microorganisms, chlorine removal occurred primarily from the meta and para positions. Dechlorination of both Aroclors was more extensive by RR microorganisms than by SL microorganisms. Consistent with these results, RR dechlorination products had less inhibitory effects on fertilization than did SL products at both concentrations tested. Thus, we conclude that microbial dechlorination of PCBs decreases or eliminates the inhibitory effects of Aroclors 1242 and 1254 on in vitro fertilization and that chlorines in the meta and para positions of biphenyl contribute significantly to such toxic effects. Also the toxicity of Aroclors, and hence the risks they pose, may be substantially reduced at sites where the PCBs have been microbially dechlorinated.

Acknowledgments This research was supported by National Institute of Environmental Health Sciences, the Michigan State Institute of Environmental Toxicology, and the Michigan Agricultural Experimental Station.

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Literature Cited (1) Hutzinger, O.; Veerkamp, W. In Microbial Degradation of Xenobiotics and Recalcitrant Compounds; Leisinger, T., Hutter, R., Cook, Am., Nuesch, J., Eds.; Academic Press: New York, 1981; p 3.

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Received for review December 13, 1995. Revised manuscript received March 7, 1996. Accepted March 11, 1996. ES950929X