Environ. Sci. Technol. 1992, 26, 1540-1544
Sittig, M. Pesticide Manufacturing and Toxic Materials Control Encyclopedia; Noyes Data Corp.: Park Ridge, NJ, 1980.
Cochrane, W. P.; Forbes, M.; Chau, A. S. Y. J. Assoc. Off. Anal. Chem. 1970,53, 769-774. Gab, S.; Born, L.; Parlar, H.; Korte, F. J. Agn'c. Food Chem.
(31) Venema, A.; Henderiks, H.; Geest, R. v. J . High Resolut. Chromatogr. 1991, 14, 676-680. (32) March, J. Advanced Organic Chemistry, 3rd ed.; John Wiley and Sons: New York, 1985; pp 82-140. (33) Buser, H. R.; Muller, M. D.,submitted for publication in Anal. Chem.
1977,25, 1365-1371.
Wilson, N. K.; Sovocool, G. W. Org. Magn. Reson. 1977, 9, 536-542.
Received for review February 10, 1992. Revised manuscript received March 30, 1992. Accepted April 8, 1992.
Biodegradation of Styrene in Samples of Natural Environments Mln Hong Fu and Martln Alexander* Laboratory of Soil Microbiology, Department of Soil, Crop, and Atmospheric Sciences, Cornel1 University, Ithaca, New York 14853
Measurements were made of the fate of styrene, a major industrial chemical, in environmental samples. The compound volatilized rapidly from shallow layers of lake water, 50% being lost in 1-3 h, but only 26% was volatilized from a 1.5-cm depth of soil in 31 days. Mineralization did not occur in sterile environmental samples. The rate of microbial mineralization was rapid in sewage, mineral soil of pH 7.23, and an organic soil; slower in groundwater and lake water; and lowest in aquifer sand, waterlogged soil, and a mineral soil of pH 4.87. The initial rates of biodegradation were linear, sometimes after an acclimation period. In 30 h, 79% or more of the styrene was sorbed to samples of mineral and organic soils, but mineralization was still rapid, suggesting that sorption is not necessarily a major limitation to the microbial transformation. The percentages mineralized per hour in soil were not greatly different at concentrations of 5.0 pg/kg to 1.0 mg/kg but were less at higher concentrations. In contrast, the percentage mineralized at 2.5,10, and 100 pg/L in lake water and at 20 and 100 hg/kg of aquifer sand was less at the lower than the higher concentrations, suggesting the existence of a threshold at still lower levels. We suggest that styrene will be rapidly destroyed by biodegradation in most aerobic environments, but the rate may be slow at low concentrations in aquifers and lake waters and in environments at low pH. Introduction Styrene is widely used for the manufacture of polystyrene, plastics, rubber, resins, and insulators; it is a common cross-linking agent in glass fiber-reinforced, unsaturated polyester resins employed in construction materials and for making small boats; and it also is a solvent for the processing of polymers. In 1990,3.64 X lo9 kg of styrene was manufactured in the United States (1). In addition to the industrial synthesis, microorganisms may form styrene from cinnamic, p-hydroxycinnamic, pcoumaric (2,3),ferulic, and caffeic acids ( 4 , 5 ) . Although much of the industrially produced styrene lost to the environment is released to the atmosphere, large quantities still enter waters and soils (6). Therefore, information on its fate in these environments is important. Scant attention has been given to the biodegradation in natural or polluted environments of this widely used compound. However, Sielicki et al. (7) reported that 95 and 87% of [8-14C]styrenewas converted to I4CO2in 16 weeks in a landfill soil and a sandy loam, respectively. A number of aerobic bacteria and fungi have been shown to be able to grow on styrene (8, 9). GrbiE-GaliE et al. (IO) showed the anaerobic transformation of styrene by a methanogenic consortium and by Enterobacter cloacae. 1540
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In the investigations with enrichment and pure cultures, a number of products formed from styrene under aerobic (7-9) and anaerobic conditions (10) have been identified. Although the metabolism of styrene has been studied with enrichment cultures or individual isolates, little information is available on the degradation of the compound in natural or polluted environments. The present investigation was therefore designed to study the fate of styrene in selected environmental samples and to assess the possible significance of volatilization, biodegradation, and sorption. Materials and Methods Reagent-grade styrene was purchased from Aldrich Chemical Co. (Milwaukee, WI). Methanol was obtained from Fisher Scientific Co., Fairlawn, NJ. [U-ringJ4C]Styrene with a specific activity of 0.66 mCi/mmol (radiochemical purity of 99%) was purchased from NEN Research Products, Boston, MA. Radiochemical purity was determined by gas chromatography. Distilled water was pasaed through the Milli-Q reagent-grade water system (Millipore Corp., Bedford, MA) before use. Samples of water were taken from Beebe Lake, Ithaca, NY, and used within 2 h of collection. The pH value of the water was 7.5, and it contained 50-60 mg of organic matter/L. Groundwater (pH 8.25, 30.5 mg of organic matter/L) containing traces of aquifer solids was obtained from Freeville, NY, and stored at 4 "C before use. Sewage (pH 7.0-7.4) obtained from the primary settling tanks of the Ithaca, NY, sewage treatment plant was used within 1day of collection. Aquifer sand (pH 6.95,0.4% organic matter) was collected from Freeville, NY, at a depth of 6.0 m. Samples of Lima loam (pH 7.23,7.5% organic matter) and Kendaia loam (pH 7.46, 5.30% organic matter) were collected from Aurora, NY; Erie silt loam (pH 4.87, 5.74% organic matter) was collected from Mt. Pleasant, NY; and Edwards muck (pH 7.50, 32.9% organic matter) was collected from a marsh in Newfield, NY. Before use, the soil samples were air-dried and passed through a 2.0-mm sieve. For experiments involving sterile environmental samples, the water and sewage were autoclaved for 25 min, the aquifer sand was autoclaved for 30 min on two consecutive days, and soil samples were irradiated with 6oCo (2.5 Mrad) . To measure mineralization, duplicate 50-g samples of soils or aquifer solids or duplicate 50-mL samples of sewage, groundwater, or lake water were amended with 25 OOO or 50OOO dpm[14C]styreneand sufficient unlabeled styrene to achieve the desired concentrations. The amended environmental samples were contained in the main compartment of 250-mL biometer flasks (Bellco Glass Inc.,
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100
0
30 VOLATILIZED
W
! i
3 i 20 W
80
f
W N
i
MINERALIZED
10
P0 d 7
c
0
10
20
30
40
0
50
0
HOURS Flgure 1. Disappearance from water samples of styrene added at a concentration of 4 mg/L.
Vineland, NJ). The side arm of the flasks contained 2.2 mL of 0.5 M NaOH to trap the 14C02produced. The soils were maintained at approximately 1bar moisture tension except for waterlogged soils, for which the soi1:water ratio was 1:2 (w/w). All samples were incubated at 22 f 2 "C in the dark. At regular intervals, NaOH was removed and replaced with fresh alkali. The NaOH removed was mixed with 3.5 mL of Liquiscint scintillation fluid (National Diagnostic, Sommerville, NJ) in 7-mL scintillation vials, and the radioactivity was counted with a liquid scintillation counter (Model LS 7500; Beckman Instruments, Inc., Irvine, CA). Loss of styrene by volatilization from the test system was avoided because the flasks were sealed. Abiotic changes were assessed with sterile environmental samples. The volatilization of styrene from deionized water and samples of Beebe Lake water was evaluated in flasks open to the air. The concentration remaining with time was determined by measuring loss of UV absorbency at 239 nm. Volatilization of styrene from soil was estimated separately using the same experimental setup as in mineralization studies, but the NaOH trapping solution in the side arm of the biometer flask was replaced with methanol to trap volatilized styrene. For studies of sorption, 25 g of sterile soil or aquifer solids was mixed with 100 mL of sterile deionized water in a 125-mL narrow-mouth amber bottle. Sterile labeled styrene (50000 dpm) and enough unlabeled styrene to achieve the desired final concentration was added aseptically. The samples were incubated at 22 f 2 "C on a reciprocal shaker operating at 45 strokes/min. At selected time intervals, the soil particles were allowed to settle, and portions of the aqueous phase were passed through sterile 0.22-pm pore-size nylon syringe filters (MSI, Westboro, MA). The filtrate then was analyzed for radioactivity. Results When added to give 4 mg/L, styrene was rapidly lost both from deionized water and water samples from Beebe Lake (Figure 1). The rate of styrene loss decreased with time, and by 40 h, no UV-absorbing compounds could be detected in solution. Tests with solutions containing 2-10 mg/L showed that about 50% of the styrene had disappeared from lake water and deionized water in 1-3 and 6-7 h, respectively. Measurements were also made of styrene volatilization from Kendaia loam contained in sealed biometer flasks. The extent of volatilization was assessed using a methanol
20
10
30
DAYS Flgure 2. Mineralizationand volatilization of styrene in Kendaia loam.
60 0 W
i A
2 40
1
/.@@+
GROUND WATER
W
10 ae 20
WATERLOGGED AQUIFER SAND
0 0
10
20
30
DAYS Flgure 3. Mineralization of styrene added at 1.0 mg/kg or 1.0 mg/L in samples from several environments.
trap, and the 14C02produced by mineralization was simultaneously determined with NaOH in parallel studies. Previous tests indicated that detectable amounts of volatilized styrene were not trapped by NaOH and that detectable quantities of 14C02were not trapped by methanol. The loam received 2 mg of the compound per kilogram of soil. Styrene was simultaneously volatilized from the soil and converted microbiologically to C02 (Figure 2). Under the test conditions, which involved use of soil in layers 1.5 cm deep, volatilization was more rapid and extensive than mineralization. Approximately 26 % was volatilized and about 13% was mineralized in 31 days. Similar results were obtained using Lima loam. As expected, the percentage volatilized was thus much lower in soils than in lake water samples at the same concentrations. An investigation was conducted to assess mineralization in samples of different environments. Lima loam and aquifer sand were amended with 1 mg of styrene/kg, and samples of sewage, groundwater, and lake water received styrene at 1mg/L. Both the rate and the extent of mineralization varied appreciably in samples taken from the several environments (Figure 3). The rates were greatest in Lima loam at a moisture tension of approximately 1bar, and in sewage, and the transformation was essentially linear with time until more than 30% had been converted Environ. Sci. Technoi., Vol. 26, No. 8, 1992
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Table I. Effect of Styrene Concentration on Its Degradation in Lima Loam
60
concn,
a
mg/ kg of soil
W N
0.005 0.020 0.10 1.0 5.0 10 50 100 500
i a
6
f
40 10 mgikg
lo00 4000
70 mineralized in 30 days
rate, rg kg-' h-l
per h
0.016 0.083 0.32 2.2 4.3 7.6 39 63 199 320 1700
0.32 0.41 0.32 0.22 0.087 0.076 0.077 0.063 0.040 0.032 0.043
55 62 50 47 31 27 26 20 16 16 17
A
100
1gll
20
0
10
20
30
DAYS Flgure 4. Mineralization of styrene at several concentrations in Lima loam.
10
to C 0 2 ;more than half of the C was mineralized in 33 days. An acclimation period of several days duration and lower rates and extents of mineralization characterized the conversion in groundwater, lake water, and waterlogged Lima loam. The rates and extents were lowest in aquifer sand, and only 10% of the compound was metabolized to C02 in about 33 days. 14C02was not formed in 33 days when labeled styrene was added to sterilized samples from the same environments. A study was conducted to evaluate the effect of styrene concentrations on the rate of its mineralization in Lima loam maintained at a moisture tension of 1 bar. Eleven concentrations were used at levels extending from 5 pg to 4.0 g per kilogram of soil. Biodegradation occurred at all concentrations, but the percentage of styrene C converted to COz in 30 days generally decreased with increasing styrene concentrations and ranged from 62% for 20 pg/kg to 16% for lo00 mg/kg (Figure 4). Only five concentrations are plotted in the figure. At concentrations greater than 100 mg/kg, mineralization began after a short acclimation period and then proceeded rapidly, and the rapid conversion was followed by a period of slow formation of 14C02 At concentrations below 100 mg/kg, biodegradation began with no apparent lag period. Mineralization at low concentrationswas initially rapid and linear, and this phase was followed by a slow release of 14C02. The relationship between concentration and the rate of degradation and percentage mineralized is shown in Table I. The rates were calculated from the activity at 0-5 days at concentrations of 0.005-1.0 mg/kg and 5-14 days at higher concentrations, which excluded the acclimation periods and nonlinear portions of the curves. The absolute rate of mineralization increased with increasing concentration. On a percentage basis, however, the rates at levels of 1.0 mg/kg or lower were not greatly affected by initial concentrations. The similarity in percentage mineralized per hour at 0.005,0.020,0.10, and 1.0 mg/kg of soil shows that the rate is directly correlated with concentration. The percentage mineralized in 30 days declined with increasing concentrations. The effect of low concentration was quite different in samples of lake water. On a percentage basis, the rate was not independent of concentration at 2.5,10, and 100 pg/L but rather diminished as the concentration fell (Figure 5A). Thus, the rate was less at the lower concentrations than 1542
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E
d
E O
3
I
B
-
0
z
1.5
100
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1.0
0.5
0 0
20
10
30
DAYS
Figure 5. Effect of styrene concentration on its mineralization In lakewater samples (A) and aquifer sand (B).
would be predicted if it is assumed that the absolute rate is a direct function of substrate level. These data suggest the existence of a threshold at a level somewhat below 2.5 pg/L. It is of interest that the percentage mineralized also diminished with decreasing concentrations. The conversion in aquifer sand was only measured at 20 and 100 pg/kg. The transformation in samples of this environment was particularly slow. Only 1.09 and 1.51% of the styrene added at 20 and 100 pg/kg, respectively, were mineralized in the test period (Figure 5B). In this instance, as in lake water, the rate at the lower level is less than would be predicted if it is assumed that the rate is a direct function of concentration. However, the extent of mineralization was only slightly greater than the impurity level of the labeled styrene. A study was conducted to compare the biodegradation in soils with a high content of organic matter (Edwards muck) and a low pH (Erie silt loam) with a soil of lower content of organic matter and near-neutral pH (Lima loam). Degradation occurred in the three soils, but the rates and extents differed (Figure 6). The acclimation period before the onset of rapid mineralization was short
I
0
10
20
30
DAYS Figure 6. Mineralization of styrene (1 mg/kg) in two mineral soils and an organic soil.
Table 11. Styrene Sorbed in Aqueous Suspensions of Environmental Samples initial concn, mg/L
1.0 10 100
% sorbed
sample
6h
30h
78h
aquifer sand Lima loam Edwards muck aquifer sand Lima loam Edwards muck aquifer sand Lima loam Edwards muck
68.8 71.9 92.5 57.1 64.2 84.0 71.6 91.5 91.2
74.6 81.4 93.9 77.7 79.2 90.4 83.9 95.9 96.3
87.4 95.1 95.5 85.1 85.1 92.7 86.7 93.3 93.2
in Lima loam and the muck soil, but this phase was prolonged and the extent of mineralization was low in the acid Erie silt loam. In no instance was C02formed from styrene in sterile soil. The rapid conversion in the muck soil suggests that hydrophobic sorption in organic matter-rich soils does not appreciably affect the microbial transformation, but the slow biodegradation in Erie silt loam may reflect a susceptibility of the active populations to low pH. Although the log KO,of styrene is only 2.87 ( I I ) , some hydrophobic sorption to the organic fraction of soils might be expected (12). Because such sorption often reduces the availability of organic substrates to microorganisms, a study of styrene sorption was conducted. For this purpose, aqueous suspensions of sterilized samples of aquifer sand, Lima loam, and Edwards muck were amended to give 1.0, 10, and 100 mg/L of the chemical, and the amount sorbed was measured at 6, 30, and 78 h. Binding was extensive even after 6 h, and the amount sorbed by the sand and loam increased with time at all test concentrations (Table 11). The percentage sorbed by the muck soil in 30 h exceeded 90% at all three concentrations. Despite the high degree of sorption, the compound was extensively mineralized in these soils.
Discussion Styrene was rapidly lost from shallow samples of lake water exposed to the air. Although volatilization thus may be a major route of styrene loss from surficial waters, volatilization from deep waters and groundwater is probably slow. Based on assumed environmental conditions, the half-life for styrene loss from surface waters has been
calculated to be 0.75 h to 51 days (13). The data presented here are within the calculated range. Volatilization was much slower in soils than in the shallow water samples, but the rate of styrene volatilization would be slower with increasing soil depth. Anderson et al. (14) compared the disappearance of several chemicals from soils as a result of volatilization and biological mechanisms. The data in the present investigation suggest that biodegradation may account for more styrene loss below the soil surface than volatilization if conditions are suitable for rapid microbial activity. Because abiotic mineralization did not occur, the mineralization in samples of all environments tested resulted from microbial activity. The rate and extent of mineralization varied with the type of environmental sample. Extensive degradation in sewage and some soils was observed when the samples presumably were aerobic. Aerobic microorganisms may convert styrene to phenylethanol, phenylacetic acid, 2-hydroxyphenylaceticacid, and styrene oxide (7, B), but these presumably are then further degraded. Mineralization was less extensive under waterlogged conditions, but the lack of O2may have resulted in the accumulation of organic products. Anaerobic enrichment cultures of bacteria have been shown to convert styrene to a series of aromatic, alicyclic, and aliphatic products (IO),and these probably persist. Mineralization of styrene at a range of concentrations was determined at both environmentally realistic low concentrations and the high levels that may exist at some disposal sites. The finding that the rate of mineralization was directly proportional to concentration at 1.0 mg/kg and below suggests first-order kinetics, but the rate was less than directly proportional above 1.0 mg/kg of soil. A threshold concentration sometimes exists below which an organic compound may not support microbial growth, and hence the compound may not be biodegraded and may persist if the initial population density is low (15). The finding that the percentage mineralized per hour decreased in lake water and aquifer sand with decreasing styrene concentrations is consistent with the view that a threshold exists in these environments, but that putative threshold must be below the concentrations tested. No evidence for a threshold was found in soil, which is consistent with determinations made of several other compounds (16). The diminution in the extent of mineralization in lake water with decreasing substrate concentrations differs from observations on the transformation of benzoate, phenylacetate, and p-nitrophenol (17). The rates and extents of mineralization were markedly different among the soils. The low pH of Erie silt loam could be the reason for the low rate of biodegradation. Although sorption to the solids was appreciable and the degree of sorption was related to the organic matter content of the samples, the compound was extensively mineralized even in the muck soil. The lack of appreciable effect of sorption on the rate of degradation may have resulted from the direct use by microorganisms of styrene retained on the surfaces, the capacity of microorganisms to excrete metabolites that promote desorption, or the spontaneous desorption of the compound. Registry No. Styrene, 100-42-5.
Literature Cited (1) Relsch, M.S. Chem. Eng. News 1991,69 (14),13-19. (2) Chen, S. L.;Peppler, H. J. J.Biol. Chem. 1956,221,101-106. (3) Clifford, D.R.;Faulkner, J. K.; Walker, J. R. L.; Woodcock, D.Phytochemistry 1969,8,549-552. (4) Finkle, B.J.; Lewis, J. C.; Corse, J. W.; Lundin, R. E. J. Biol. Chem. 1962,237,2926-2931. Environ. Sci. Technol., Vol. 26, No. 8, 1992
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( 5 ) Finkle, B. J. Nature (London) 1965,207, 604-605. (6) Public Data Branch, Office of Toxic Substances, U.S. Environmental Protection Agency, Washington, DC, 1991. (9Sielicki, M.; Focht, D. D.; Martin, J. P. Appl. Environ. Microbiol. 1978, 35, 124-128. (8) Baggi, G.; Boga, M. M.; Catelani, D.; Galli, E.; Treccani, V. Syst. Appl. Microbiol. 1983, 4, 141-147. (9) Hartmans, S.; van der Werf, M. J.; de Bont, J. A. M. Appl. Enuiron. Microbiol. 1990, 56, 1347-1351. (10) GrbiE-GaliE, D.; Churchman-Eisel, N.; MrakoviE, I. J.Appl. Bacteriol. 1990, 69, 247-260. (11) Banerjee, S.; Howard, P. H. Environ. Sci. Technol. 1988, 22, 839-841. (12) Hassett, J. J.; Banwart, W. L. In Reactions and Movement of Organic Chemicals in Soils; Sawhney, B. L., Brown, K., Eds.; Soil Science Society of America: Madison, WI, 1989; pp 31-44.
(13) Environmental Protection Agency. Occurrence of Synthetic Organic Chemicals in Drinking Water, Food, and Air; Office of Drinking Water, U.S. Environmental Protection Agency: Washington, DC, 1987. (14) Anderson, T. A.; Beauchamp, J. J.; Walton, B. T. J. Environ. Qual. 1991, 20, 420-424. (15) Alexander, M. Environ. Sci. Technol. 1985, 18, 106-111. (16) Scow, K. M.; Simkins, S.; Alexander, M. Appl. Environ. Microbiol. 1986, 51, 1028-1035. (17) Rubin, H. E.; Subba-Rao, R. V.; Alexander, M. Appl. Environ. Microbiol. 1982, 43, 1133-1138. Received for review January 29, 1992. Revised manuscript received April 17,1992. Accepted April 20,1992. Support for this research was provided by the Styrene Information and Research Center.
A History of Octachlorodibenzo-p-dioxin, 2,3,7,8-Tetrachlorodibenzofuran, and 3,3',4,4'-Tetrachlorobiphenyl Contamination in Howe Sound, British Columbia Roble W. Macdonald," Waiter J. Cretney, Norman Crewe, and David Paton Institute of Ocean Sciences, P.O. Box 6000, Sidney, British Columbia, V8L 462 Canada --
Six pulp mills using chlorine bleach discharge effluent into the Strait of Georgia, British Columbia. Fisheries were closed around the mills when dioxins and furans were measured in sediments and edible seafood. We report here the first measurements made for a dioxin (OCDD), a furan (2,3,7,8-TCDF),and a PCB (77) on three dated box cores collected from the region. OCDD is widely distributed, first appears in the sediments in 1940, reaches a maximum in 1970, and probably has an atmospheric source. In contrast, 2,3,7,8-TCDF is concentrated near pulp mills and has been accumulating in the sediments since -1965. PCB 77 correlates well with OCDD and is probably supplied from the atmosphere. ii
-
Introduction
P ~ d pmills using chlorine produce dioxins and furans (1, ?,!. British Columbia has 10 mills that discharge chlori-
nated effluent directly to the marine environment, and surface sediments near them are found to be contaminated with organochlorines (3). Two pulp mills are situated on Howe Sound and another four are located on the Strait of Georgia (Figure 1). Howe Sound has long received contaminant inputs ( 4 ) . Since 1899, Cu and Zn have entered the inlet from a mine at Britannia either as tailings or in acid drainage. From -1965 to 1970, a Hg cathode chlor-alkali plant (FMC) disposed -20 kg of Hg/day in effluent forcing closure of shellfkh and groundfish fisheries for 8 yr (5). Due to furan and dioxin contamination, in 1988 (November) harvesting of prawn, shrimp, and crab was curtailed near pulp mills; more extensive closures have followed. Pulp mill effluent has been reviewed as a coastal contaminant (6, 7), but byproduct organochlorines have gone unmentioned. Only recently have we been able to measure these compounds at the ultratrace concentrations which, we are now realizing, are of significance to human health and the ecosystem. For the West Coast there are few data for dioxins or furans, none reported in the open literature (3, 8-10). 1544
Environ. Sci. Technol., Vol. 26, No. 8, 1992
We report here octachlorodibenzo-p-dioxin (OCDD), 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-TCDF), and 3,3',4,4'-tetrachlorobiphenyl (PCB 77) determinations performed on sediments from three box cores dated by 210Pbgeochronology. We focus on these toxic, planar compounds because they represent the various compound classes and because they represent various expected sources. OCDD is known to have an important atmospheric source (11-13) while 2,3,7,8-TCDF is primarily associated with pulp mills (1-3,14). PCB 77 was measurable by making a small alteration to the dioxin procedure. Such non-ortho-substituted (coplanar) PCBs (15) rank with furans and dioxins in toxicity (16,17), and yet we have few data on their sources and budgets. We attempt to establish the source, the history, the distance of transport, and the magnitude of burdens and fluxes of these three contaminants to the sediments of Howe Sound and the Strait of Georgia. Such information is crucial to understand the environmental role of pulp mills, to design appropriate monitoring strategies, and to predict the effect of remedial action taken by pulp mills. S a m p l i n g and S u b s a m p l i n g M e t h o d s
In December 1990, two box cores (TC-1, HS-1) were collected in Howe Sound (Figure 1)and one was collected from Ballenas Basin (BB-1). Ballenas Basin was chosen partly because previous work has established geochronology (18) and partly to provide a basin-scale backdrop. Upon retrieval of each box core (0.06 m2,0.5 m/s), one wall of the stainless steel liner was lowered in the ship's laboratory to subsample each core with minimal disturbance. Stainless steel tools were cleaned between samples (tap water, distilled water, acetone), and sediment from the outer 5 cm of the box was discarded. Subsamples (I-cm interval for top 10 cm and 2-cm intervals deeper) were split for organochlorines (glass Mason jars with Teflon-lined lids) and other determinations (Whirl-pak bags) and frozen. Mason jars were stringently precleaned (detergent rinse, 4-h soak in 2% RBS, rinse, distilled water
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