Environ. Sci. Techno/. 1995, 29, 702-708
Gemtoxicity M l e s and Reaction Chcteristics of Potassium P ne Glycol Dehalsgenartik of -Wood Presedng Waste M A R J O R I E S . H O N G , LINGYU H E , BRUCE E . D A L E , A N D K I R B Y C . DONNELLY* Department of Chemical Engineering, Toxicology Program, Texas A&M University, College Station, Texas 77843
Chemical dehalogenation of pentachlorophenolcontaining wood preserving waste (EPA K001) with potassium polyethylene glycol (KPEG) was investigated. The effect of treatment on the waste toxicity was examined by monitoring genotoxicity of the organic wastes. The effects of temperature, reaction time, and pentachlorophenol concentration on dehalogenation were also investigated. Samples were collected throughout treatment and sequentially extracted with dichloromethane and methanol. Chemical analysis indicated that KPEG effectively dehalogenated pentachlorophenol in the waste. Mutagenicity and genotoxicity of the organic extracts were evaluated in the SalmonelMmicrosome and Escherichia coli prophage induction assays. Extracts exhibited significantly higher genotoxic responses when tested with metabolic activation. KPEG dehalogenation generally decreases waste genotoxicity during treatment; however, additional treatment is required for complete detoxification.
Introduction Halogenated hydrocarbons, particularly halogenated aromatic hydrocarbons such as polychlorinated biphenyls (PCBs),pentachlorophenol (PCP),polychlorinated dibenzop-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs),are persistent environmental contaminants that have been detected throughout the global ecosystem (13). These compounds are known to cause serious toxicologicaleffects (PCP,refs4-16; PCBs, refs 17-21; PCDDs, refs 22 and 23). Halogenated aromatics represent significant environmental and human health hazards. In the past decade, dehydrohalogenation reactions have been investigated as an alternative treatment method for halogenated organics (24-29). However, this chemical method is usually not effective for lower chlorinated hydrocarbons unless the reaction is carried out at an extremely high temperature. Biodegradation of halogenated wastes has also been studied extensively (30,31)but is usually less effective when the more toxic higher chlorinated hydrocarbons are present (30,32-38). Also, a frequent problem in complex mixture biodegradation is the increased toxicity of remediated products (3941).
An assessment in 1980 (42) reported wood preserving waste 0 generation to be approximately 4.4 million lb/year. These complex wastes may contain hundreds of compounds, including phenolic and aromatic hydrocarbons, polynuclear aromatic hydrocarbons, chlorophenols, PCDDs, and PCDFs. Because of the complex nature of WPW, remediation of contaminated sites is difficult. The WPW containing PCP served as the model waste for KPEG dehalogenation in this research. The environmental fate and toxicologicalproperties of the products of KPEG chemical treatment have not been described by the available data. However, previous studies (24, 27, 29) strongly recommended that the toxicological characteristics of reaction products resulting from KPEG treatment be described. Determining the toxicity profiles of degradation treatments is necessary to establish whether the loss of the parent compound reduces toxicity or completely detoxifies the hazardous waste. The toxicity of hazardous waste-before, during, and after various remediation processes-is not currently a design parameter in establishing remediation technologies. In fact, this aspect of remediation feasibilitystudies is often overlooked; many of such studies focus mainly on waste degradation and seldom investigate waste detoxification. Degradation of hazardous waste does not necessarily provide detoxification. Certain remediation methods may yield products that are actuallymore toxic than the starting materials. Also, without monitoring toxicity throughout the remediation process, the potential effects of toxicity changes on the surrounding environment cannot be assessed. These facts strongly suggest that toxicological profiles of a hazardous waste during remediation must be measured to establish the potential of that technology to reduce toxicity. This research attempts to elucidate the effects of * Corresponding author; e-mail address:
[email protected].
702 ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 3,1995
0013-936X/95/0929-0702509.00/0
@ 1995 American Chemical Society
the KPEG treatment method on the toxicity of a real waste by monitoring genotoxicity of the organic extracts of the waste.
Materials and Methods KPEG Reagent. The KPEG reagent was synthesized from potassium hydroxide pellets (85%)and polyethyleneglycol monomethyl ether (molecular mass 350 Da) (24). Excess potassium hydroxide was added to polyethylene glycol (molar ratio 1.5:l). The KOH pellets and PEG were heated under nitrogen at approximately 100 "C. Nitrogen flow was provided to facilitate water removal. Contents of the beaker were stirred for 45 min or until most of the pellets were dissolved. The KPEG reagentwas stored in a desiccator until required for the KPEG treatment experiments. Waste. Wood preserving bottom sediment EPA K001, which contains both PCP and creosote, was the model waste in the KPEG dehalogenation study. The hygroscopic nature of polyethylene glycol reduces the reactivity of KPEG in the presence of water. Hence, the waste was dried in an oven at 120 "C to remove water prior to KPEG treatment. The heterogeneous WPW waste was mixed thoroughly prior to both drying and chemical treatment to maintain its properties from experiment to experiment. KPEG Chemical Treatment. Four test temperatures were selected for isothermal KPEG dehalogenation studies: 30, 48, 80, and 120 "C. KPEG treatments were performed twice at each temperature for a duration of 30 min each. Samples were collected at 2, 5, 10, and 30 min during the treatment. Equal masses of dried WPW and KPEG reagent were separately brought to the reaction temperature on a hot plate. KPEG was then added to the WPW in a high-strength beaker, and the mixture was mechanically stirred during the 30-min treatment. At the selected sampling times (2, 5, 10, and 30 min), 50-g samples of the reaction mixture were collected in glass jars containing 15 mL of 50%solution of sulfuric acid. The acid-waste mixture was stirred immediately to quench the reaction. The acid volume required for the quench was calculated based on the assumption that KPEG did not react with waste components. Because sampling was performed manually, the zero-time samples were handled prior to the treatment run. KPEG (25 g) was neutralized by the acid in the sampling jar and mixed with 25 g of the dried WPW. Samples were stored in the laboratory cold-room at 4 "C. Extraction Procedure and Chemical Analysis. The neutralized WPW samples (pH 7 ) collected throughout the KPEG treatment period were sequentially extracted with dichloromethane and methanol by employing the Tecator Soxtecprocedure (43). The extraction procedure also served to reduce microbial contamination problems that might occur in subsequent bioassays by removing indigenous microbial populations that may have been present in the waste. PCP concentrations in the residues from the extraction were analyzed with a gas chromatograph (GC) equipped with a 63Nielectron capture detector (ECD)and Megabore column DB5-MS (30 m x 0.53 pm i.d., 1.5 mm 0.d.) (JMW Scientific, Fulsom, CA). The following temperature/time sequence was entered for PCP analysis: the column temperature was held at 100 "C for 3 min, increased at 5 "Clmin until the temperature reached 250 "C, and finally held at 250 "C for 5 min. The column was routinely cleared
of heavy compounds from previously injected WPW extracts by maintaining the column temperature at 250 "C for 10 h. SalmonellalMicrosome Assay. The Salmonellulmicrosome assay (44, 45) was performed for mutagenicity analysis of the waste extracts. Salmonella typhimurium tester strain TA98 was supplied by B. N. Ames (University of California, Berkeley). The extract residues of the treated waste were resuspended in dimethyl sulfoxide (DMSO)and tested on duplicate plates in two independent experiments at five dose levels (0.05,0.10,0.25,0.50,and 1.0 mglplate). The extracts were tested in the plate-incorporation assay in the presence and absence of metabolic activation (S9 mixture prepared with S9 fraction of Aroclor 1254-induced Sprague-Dawley rat liver). The first Salmonella assay performed was with extracts from 80 "C Run A, which was tested with a lower level of metabolic activation (20% S9 mix) than the subsequent runs. Because responses were higher with 30%S9 mix, the remaining runs were performed with 30% S9. Escherichia coli Prophage Induction Assay. Genotoxicity of chemicals in the waste extracts was also evaluated in the E. coli prophage induction assay (12, 46). Tester strains, E. coli lysogen WP24 and indicator TH008, were provided by D. M. DeMarini (US. EPA, Research Triangle Park, NC). Extract residues were resuspended in acetone and tested both with and without metabolicactivation (2.5% S9 mix). The mass ratio of S9 fraction to extract in both the Salmonellalmicrosome and E. coli prophage induction assays was the same. The extract residues were tested in duplicate in at least two independent experiments. The criteria for positive response ratings were based on established criteria for this bioassay (47, 48). For each independent experiment, test compounds were scored as positive (+) if they induced at least a 3-fold increase of induced plaque-forming unitslplate over that induced by the corresponding acetone control at two or more doses in one experiment; test compounds were scored as weak positive (w+)if they reached or exceeded a 3-fold increase at only one dose. A summary response was given to each tested extract based on the reproducibility of the results from two independent experiments. If a given response was not reproducible, the response summary was scored as questionable (?).
Results and Discussion The results presented here include: (1)KPEG reduction of PCP in WPW, (2) waste genotoxicity profiles with respect to KPEG treatment time and bioassay dose (Salmonella/ microsome bioassay measurements), and (3) waste genotoxicity as measured with the E. coli prophage induction assay. PCP Reduction. The percent PCP reduction was estimated for each treatment. The percent PCP reduction achieved with the KPEG reagent was defined as ([PCPlinitid - [PCPlfin31[PCPl,nitialx 100. Figure 1compares the percent PCP reductions achieved during KPEG treatments performed isothermally at 30,48, 80, and 120 "C. This figure shows that the percent PCP reduction increases with increasing temperature, indicating that the reaction is endothermic. Mutagenicity Profiles of KPEG Treatment of WPW As Measured by SalmonellalMicrosome Assay. The normalized mutagenic potential is defined as the ratio of the number of revertantdplate formed from the test compound VOL. 29, NO. 3, 1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY
703
,
1uu
30'C
I
'I0
h
E
80 -
48'C
m
c
.? U a a
60 -
U
40 -
0)
& k
u
20
tL
0
-
5
10 I5 20 25 30
0
5
Time ( m i n )
BOT
0
30 (5 min)
30
48
80
10 15 10 I S 30
Time (mln)
120°C
120
Temperature ("C) FIGURE 1. Percent PCP reduction in WPW treated with KPEG.
to those formed by the control solvent, DMSO. When the mutagenic potential has a value equal to or greater than 2 for two consecutive doses, the test compound is considered mutagenic. The extracts were not mutagenic when tested without metabolic activation in the Salmonella assay. However, the extracts were mutagenic when tested in the assay with metabolic activation. Also, the methanol extracts from the KPEG-treated WPW showed much higher levels of mutagenic activity than the dichloromethane extracts. This difference is attributed to the character of the extracts; the dichloromethanefraction consists primarily of polar neutral compounds (PCP, chlorinated contaminants, and some polynuclear aromatic hydrocarbons (PNAs)),whereas the methanol fraction consists primarily of base neutral compounds (PNAs). In several cases, the extract PCP concentration appears to correlate with the mutagenic effect. Extractswith higher PCP concentrations appeared to correlate with lower mutagenic responses. This effect may be occurring because of PCP's acute toxic effect on the Salmonella, resulting in reduced numbers of viable cells such that the mutagenic effect of the PNAs could not be observed. Mutagenic potentials indicate the mutagenicity of the organic extracts from the waste samples. Weighted mutagenic activity is an indicator of the mutagenicity of the waste with respect to a particular solvent extract (e.g., dichloromethane or methanol fraction). The specific mutagenic activity (net revertantdmg of extract) is defined as the background number of his+ revertants (mutated Salmonella colonies) caused by the DMSO solvent control subtracted from the total his+ revertants caused by 1mg/ plate of test extract. The weighted mutagenic activity (net revertantdg of waste) is defined as the product of the specific mutagenic activity (net revertantslmg of extract) and the milligrams of organics extracted per gram ofwaste. The sum of the weighted mutagenic activities of the dichloromethane and methanol extracts defines the cumulative weighted mutagenic activity or waste mutagenicity. This value is extremely important in evaluating the mutagenicity of the whole waste material. The waste mutagenicity profiles of KPEG treatments at all temperatures are compared in Figure 2. These mutagenic responses were measured in the assay with 30% S9 mix at a dose to the Salmonella of 1 mg/plate. A definite trend appears in the waste mutagenicity as a function of treatment time; the mutagenicity of the waste initially increases and subsequently decreases with reaction time. 704
4
ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 3, 1995
0
5
10 15 20 25 30
0
Time (mi")
5
10 15 1 0 25 30
Time (min)
FIGURE 2. Cumulative weighted mutagenic activity profiles of KPEG dehalogenation of WPW performed at 30, 48. 80, and 120 "C (Se/mone//a/microsome assay: 30% S9 mix, 1 mg/plate). -0-.-
3Q'C
-*-
IB'C
-A-
IZO'C
8Q'C
a
0.6
C
5
10
15
20
25
30
Time ( m i n )
FIGURE 3. Comparison of normalized cumulative weighted mutagenic activity profiles for KPEG treatment of WPW at 30, 48, 80, and 120 "C.
Figure 3 compares the normalized waste mutagenicity profiles of the same KPEG treatments of WPW shown in Figure 2. The waste mutagenicity values were normalized by dividing the waste mutagenicity at any reaction time by the initial waste mutagenicity. These results indicate that the genotoxicity of reaction intermediates from KPEG treatment at the lower temperatures of 30 and 48 "C exhibit a higher overall toxicity than at the higher temperatures of 80 and 120"C. Genotoxicityprofilesfor specific remediation technologies may permit comparison of process variable levels to choose approximate optimal conditions for treatment of hazardous wastes. E. coli Prophage Induction Assay Results. The Salmonellalmicrosome assay is not sensitive in detecting the genotoxicity of chlorinated phenols. Hence, the residues were also tested in the E. coli prophage induction assay. Tables 1and 2 show the E. coli test responses of the extracts from KPEG treatment of WPW performed at 30 (run A) and 120 "C (run B). These results indicate that the Salmonella test is a better bioassay for detecting the genotoxicity of KPEG-treated WPW than the E. coli test.
TABLE 1
Induction of Prophage A by WPW Treated with RPEG at 30 O C , Run Aa exDeriment 1 induced PFU/plate (fold increase) -s9 +s9
fraction
time (min)
dose (mg)
MeC12
WPW
0.031 0.063 0.130 0.250 0.500
* (0.9) * (0.3) * (0.4) * (0.0)
MeC12
0
0.031 0.063 0.130 0.250 0.500
MeClz
MeC12
MeC12
2
5
10
experiment 2
15 (1.4)
dose (mg)
-
41 (1.9) * (0.6) * (0.6) * (0.5) * (0.1) -
0.016 0.031 0.063 0.130 0.250
1 1 (1.3) 19 (1.7) * (0.9) * (0.1) 22 (1.2) -
56 (2.3) 1 1 (1.2) * (0.3) * (0.5) * (0.4) -
0.031 0.063 0.130 0.250 0.500
* (0.9) * (1.0)
0.031 0.063 0.130 0.250 0.500
3 (1.1) * (0.9) * (0.5) * (0.2) 21 (1.2) -
0.031 0.063 0.130 0.250 0.500
6 (1.2) * (1.0) * (0.6) * (0.1) * (0.1)
* (0.6) * (0.2) * (0.4) -
-
induced PFUlplate (fold increase) -s9 +59
13 (1.5) 13 (1.6) * (1.0) * (0.1) * (0.8) -
W+
-
0.016 0.031 0.063 0.130 0.250
14 (1.5) 7 (1.3) 16 (1.9) * (0.6) * (0.3) -
41 (2.3) * (0.8) 5 (1.1) * (0.4) * (0.2) -
-
38 (1.8) 10 (1.2) * (0.4) * (0.4) * (0.6) -
0.016 0.031 0.063 0.130 0.250
10 (1.4) 10 (1.5) 1 1 (1.6) * (0.4) * (0.2) -
* (0.9) * (0.6) * (0.5) * (0.2) -
-
95 (3.1) * (1.0) * (0.6) * (0.5) 6 (1.1)
0.016 0.031 0.063 0.130 0.250
16 (1.6) 8 (1.4) 16 (1.9) * (0.4) * (0.2) -
43 (2.2) 12 (1.2) * (0.8) * (0.4) * (0.2) -
-
0.016 0.031 0.063 0.130 0.250
4 (1.1) * (0.8) 9 (1.5) * (0.3) * (0.1)
22 (1.6) * (0.8) * (0.8) * (0.3) * (0.2) -
-
-
* (0.9)
12 (1.2) * (0.5) * (0.4) * (0.6) -
-
126 (4.6) 3 (1.1) 19 (1.4) 5 (1.1) * (0.3)
summary response -59 +59
18 (1.5)
MeC12
30
0.031 0.063 0.130 0.250 0.500
4 (1.1) * (1.0) * (0.6) * (0.3) * (0.2)
51 (2.1) 20 (1.3) * (0.6) * (0.3) * (0.5) -
0.016 0.031 0.063 0.130 0.250
20 (1.8) 13 (1.6) 1 1 (1.6) * (0.4) * (0.3)
36 (2.0) 9 (1.2) * (0.6) * (0.3) * (0.3) -
MeOH
0
0.031 0.063 0.130 0.250 0.500
29 (1.8) 42 (2.5) 35 (2.1) * (0.7) * (0.1) -
116 (3.6) 41 (1.7) 17 (1.3) * (0.6) * (0.2)
0.016 0.031 0.063 0.130 0.250
15 (1.6) * (1.0) 9 (1.5) * (0.7) * (0.8) -
89 (3.6) 45 (1.8) 9 (1.2) 2 (1.0) * (0.2)
0.031 0.063 0.130 0.250 0.500
39 (2.1) 79 (3.8) 60 (2.8) 6 (1.1) * (0.0)
113 (3.5) 122 (3.0) 52 (1.8) * (1.0) * (0.9)
0.016 0.031 0.063 0.130 0.250
14 (1.5) 29 (2.3) 17 (1.9) * (0.5) * (0.4)
67 (2.9) 51 (1.9) l(1.0) * (0.9) * (0.5) -
177 (4.9) 167 (3.7) 133 (3.1) 57 (1.9) 8 (1.2)
0.016 0.031 0.063 0.130 0.250
20 (1.8) 36 (2.7) 33 (2.9) * (0.9) l(1.0) -
?
0.016 0.031 0.063 0.130 0.250
10 (1.4) 24 (2.1) 26 (2.5) * (0.9) * (0.7) -
74 (3.1) 68 (2.2) 55 (2.0) 27 (1.5) * (0.4) w+ 84 (3.4) 48 (1.9) 52 (2.0) 48 (1.9) 31 (1.5) W+
?
MeOH
2
-
+
MeOH
5
0.031 0.063 0.130 0.250 0.500
108 (4.0) 87 (4.1) 109 (4.3) l(1.0) * (0.5)
+
+
MeOH
10
0.031 0.063 0.130 0.250 0.500
43 (2.2) 89 (4.2) 133 (5.0) 96 (2.5) * (0.6)
225 (6.0) 290 (5.7) 141 (3.2) 113 (2.9) 4 (1.1)
+
+
-
-
-
?
* MeCI2, dichloromethane (extraction solvent); MeOH, methanol (extraction solvent); WPW, untreated waste.
All of the extracts were tested at five doses starting from the first turbid well next to a clear well. However, the turbid wells frequently produced few or no plaque-forming units
(PFUs). Turbid wells forming few or no plaques, represented by fold increases in genotoxicitylessthan 1,indicated acute toxicity. VOL. 29, NO. 3,1995 /ENVIRONMENTAL SCIENCE &TECHNOLOGY 1706
TABLE 2
Induction of Prophage A by WPW Treatment with KPEG at 128 O C , Run Ba experiment 2
experiment 1
induced PFU/plate (fold increase)
induced PFU/plate (fold increase) fraction
time (min)
MeC12
WPW
MeC12
MeC12
MeC12
MeC12
MeCh
0
2
5
10
30
dose (mg)
-s9
+s9
0.031 0.063 0.130 0.250 0.500
* (0.4) * (0.8) * (0.5) * (0.5) * (0.0)
* (0.9) * (0.7) * (1.0) * (0.8) * (0.0)
0.031 0.063 0.130 0.250 0.500
* (0.4)
0.031 0.063 0.130 0.250 0.500
MeOH
MeOH
MeOH
0
2
5
10
+59
0.016 0.031 0.063 0.130 0.250
-
12 (2.2) 2 (1.2) * (0.5) 14 (2.4) 13 (1.7) -
* (0.8) 65 (1.9) 14 (1.2) * (0.7) * (0.4) -
0.016 0.031 0.063 0.130 0.250
l(l.l) 6 (1.7) 2 (1.2) 4 (1.3) * (1.0)
9 (1.9) 6 (1.4) * (0.9) 4 (1.5) * (0.6)
* (0.4) * (0.8) * (0.6) * (0.3) * (0.0)
* (0.9)
0.016 0.031 0.063 0.130 0.250
4 (1.4) l(l.l) * (0.8) 3 (1.3) -
12 (2.2) 2 (1.2) * (0.8) 3 (1.3) 2 (1.1) -
0.031 0.063 0.130 0.250 0.500
* (0.4) * (0.6)
35 (1.2) 18 (1.2) 45 (1.7) * (1.0) * (0.4) -
0.016 0.031 0.063 0.130 0.250
6 (1.5) 4 (1.5) 4 (1.4) l(1.1) * (0.9)
16 (2.7) 7 (1.5) * (0.8) 4 (1.5) * (0.8) -
0.031 0.063 0.130 0.250 0.500
* (0.4) 9 (1.1) * (0.7) * (0.3) * (0.5) -
* (1.0) 12 (1.2) 16 (1.2) 27 (1.3) * (0.5)
0.016 0.031 0.063 0.130 0.250
* (1.0) * (0.8)
22 (3.3) 16 (2.1) * (0.9) 6 (1.6) * (0.8)
0.031 0.063 0.130 0.250 0.500
* (0.6)
* (0.8)
3 (1.0) 8 (1.1) * (0.3) * (0.3)
5 (1.1) 7 (1.1) * (0.6) * (0.4) -
-
* (0.9) * (0.8) * (0.2) * (0.2) -
-
* (0.5)
* (0.3) * (0.3) -
-
4 (1.1) * (0.9) * (0.7) * (0.5) -
-
* (0.7)
-
6 (1.5) 4 (1.3) * (0.8) -
-
W+
0.016 0.031 0.063 0.130 0.250
12 (2.0) 3 (1.3) * (0.9) * (1.0) 2 (1.2) -
15 (2.5) 8 (1.6) * (0.6) 4 (1.5) * (1.0) -
28 (1.3) * (0.5) * (0.1) -
40 (1.3) 32 (1.4) 28 (1.4) 43 (1.5) * (0.6) -
0.016 0.031 0.063 0.130 0.250
4 (1.3) 8 (1.9) 6 (1.5) * (1.0) 5 (1.4) -
20 (3.0) 16 (2.1) 4 (1.2) 13 (2.3) 2 (1.1)
* (0.8)
* (0.8)
10 (1.1) 5 (1.1) 81 (1.7) * (0.6) -
36 (1.5) 50 (1.8) 96 (2.0) * (0.8) -
0.016 0.031 0.063 0.130 0.250
1 1 (2.0) 6 (1.7) 7 (1.6) 7 (1.5) 12 (1.9) -
24 (3.5) 26 (2.8) * (0.5) 20 (3.0) 3 (1.2)
0.031 0.063 0.130 0.250 0.500
* (0.6) * (0.9) * (0.9) * (0.9) * (0.5)
* (0.6) 39 (1.5) 73 (2.1) 122 (2.3) * (0.5)
0.016 0.031 0.063 0.130 0.250
14 (2.2) 12 (2.3) 1 1 (2.0) 21 (2.4) 17 (2.3) -
18 (2.8) 16 (2.1) * (0.6) 5 (1.5) * (0.8) -
0.031 0.063 0.130 0.250 0.500
* (0.5)
* (0.7)
71 (2.0) 44 (1.5) * (0.9) * (0.7)
10 (1.1) 87 (2.3) 38 (1.4) * (0.6) -
0.016 0.031 0.063 0.130 0.250
29 (3.6) 22 (3.5) 20 (2.7) 8 (1.6) 10 (1.8)
14 (2.5) 9 (1.6) 6 (1.3) 17 (2.8) 4 (1.2) -
0.031 0.063 0.130 0.250 0.500 0.031 0.063 0.130 0.250 0.500
* (0.4) * (0.8)
-
a
-s9
1 1 (2.0) 5 (1.6) 2 (1.2) * (0.5) 58 (5.1)
-
MeOH
dose (me)
-
summary response
+
Wt-
+
MeC12, dichloromethane (extraction solvent); MeOH, methanol (extraction solvent); WPW, untreated waste.
Of the 40 individual responses reported here (two experiments for each of the 20 test fractions tested with and without metabolic activation), reproducible summary 706 1 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 3,1995
responses (+, -, or Wi-) were obtained for all but nine pairs of responses. The extract test results in the assay were reasonably reproducible.
The E. coli results show that as the test fraction dose decreased, the apparent response increased. This relation is often observed with chemicals, such as benzo[a]pyrene, for which the genotoxic effect is masked by the acute toxicity effects caused by higher doses of the test compound. At mid-range to low doses, the acute toxic effects are considerably reduced, thereby allowing the detection of genotoxic responses. Chemicals with positive responses and inverse dose-response behaviors may induce a higher percentage of genetic damage in viable cells at higher doses than at lower doses. This effect would suggest that actual dose-related increases in the genotoxic responses should be occurring but are not readily observable because of reduced cell viability at the higher doses. The dramatic difference between the E. coli and Salmonella responses observed for these test compounds may occur because of the sensitivity of the E. coli assay to the chlorinated species. The interactive effects between the toxic chlorinated compounds and the PNAs in WPW overwhelmed the E. coli tester strains in the E. coli bioassay.
Conclusions This research shows the effects of KPEG treatment on waste toxicitythroughout the treatment process. KPEG treatment effectively dechlorinates PCP in WPW. Greater dechlorination was achieved in wastes treated at higher temperatures (80 and 120 "C). KPEG treatment reduced the waste genotoxicity slightly. While the waste mutagenicity appears to increase initially during treatment, this observation is due to the reduction of PCP and its acute toxic effects, which mask potential mutagenic responses. The WPW contains creosote, PCP, and PCP microcontaminants. The Salmonella assay does not detect mutagenicity of chlorinated compounds; therefore, the observed mutagenic response results from the polynuclear aromatic hydrocarbon contaminants in the waste. Donnelly (49) showed that although the mutagenicity of PCP was not detected in the Salmonellalmicrosome assay, the effect of PCP in a mixture with benzo[alpyrene tested in the assay could cause additive, synergistic, or antagonistic interactive effects;the resulting interactive effect observed was strongly dependent on the relative concentrations of the chemicals in the mixture. Hence, these results and Donnelly (49) show that KPEG reaction with PCP in the waste reduces the genotoxicity of the waste throughout treatment. Although KPEG dechlorination reduces the WPW mutagenicity, additional treatment will be required to detoxify the environmental mixture completely. KPEG selectively treats only the halogenated constituents in this waste. Biodegradation as the secondary treatment method for WPW should be examined and may be an economically feasible and appropriate method for complete detoxification of the KPEG-treated WPW. This research has also shown that short-term bioassays can be useful tools for monitoring genotoxicity of complex environmental mixtures during remediation. The Salmonellalmicrosome assay was better for detecting the genotoxicity of the WPW throughout the P E G treatment than the E. coli prophage induction assay. The sensitivity of the E. coli assay to both the acute toxic and the genotoxic effects of PCP as well as its interactive effects with other waste contaminants appear to overload the bacterial system. The Salmonella assay does not detect the mutagenicity of
chlorophenols and other chlorinated compounds; however, the acute toxic effectsof PCP and its interactive effectswith other contaminants in the KPEG-treated waste are observable in the assay. The Salmonellalmicrosome assay is an economical, rapid, and highly reproducible assay for obtaining the genotoxicity profiles during treatment of this environmental waste. Also, highlyreproducible data allow development of time-dependent genotoxicity profiles of remediation processes. These genotoxicity profiles of remediation technologies allow for process optimization with respect not only to parent compound removal but also to toxicological parameters.
Acknowledgments This research was supported in part by Grant P42-ESO4917 from the National Institute of Environmental Health Sciences. M.S.H. was supported by the National Institute of Environmental Health Sciences Toxicology Predoctoral Training Grant EHS-1742-ESO-7273-01.
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ES940390N @
Abstract published inAdvanceACSAbstracts,December 15,1994.
