Pesticides in Urban Environments - ACS Publications - American

Chapter 12

Fate of an Antifoulant in an Aquatic Environment 1

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A. Jacobson , L. S. Mazza , L. J. Lawrence , B. Lawrence , S. Jackson , and A. Kesterson Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 8, 2015 | http://pubs.acs.org Publication Date: February 18, 1993 | doi: 10.1021/bk-1993-0522.ch012

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Rohm and Haas Company, Spring House, PA 19477 PTRL-East, Inc., Richmond, KY 40475 2

RH-5287 (2-n-octyl-4,5-dichloro-1-isothiazolin-3-one) had a half-life of less than 1 hour in both an aerobic and an anaerobic aquatic microcosm consisting of marine sediment and seawater. Upon application of 0.05 ppm C RH-5287, over 90% of the C-activity partitioned rapidly into the sediment. Soxhlet extractions with dichloromethane:methanol (9:1) followed by methanol eluted approximately 30 - 60% of the total applied radioactivity. After exhaustive extraction of the post-Soxhlet extracted sediment with 0.25N HCl followed by 1N NaOH, 14 to 40% of the total applied radioactivity still remained bound to the marine sediment. In the aerobic microcosm, CO accounted for approximately 24% of the applied activity after 30 days. The production of sizable quantities of CO and extractable polar metabolites indicated that degradation involves cleavage of the isothiazolone ring. 14

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RH-5287 is a member of the 3(2H)-isothiazolone class of compounds which have demonstrated biocidal activity against a wide spectrum of bacteria, fungi and algae (1). RH-5287, when formulated as Sea-Nine 211™ Biocide, has been found to be an effective active ingredient in marine paint formulations to prevent buildup (fouling) of algae and invertebrate animals on submerged hulls of ships and other vessels. Isothiazolones are known to undergo chemical hydrolysis, especially in the presence of a nucleophile (2,3). Previous studies on other 3(2H)-isothiazolones has shown that biological degradation of these compounds is very rapid (4). The metabolic pathway involves cleavage of the isothiazolone ring and subsequent oxidation of the terminal methylene substituents.

0097-6156/93/0522-0127$06.00/0 © 1993 American Chemical Society

In Pesticides in Urban Environments; Racke, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

PESTICIDES IN URBAN ENVIRONMENTS

128

In order to assess the safety and evaluate the impact of RH-5287 in the marine environment we performed an aerobic and an anaerobic microcosm study. The microcosms, consisting of marine sediment and seawater, were monitored for volatiles, degradation kinetics, partitioning between seawater and sediment, and degradation products.

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 8, 2015 | http://pubs.acs.org Publication Date: February 18, 1993 | doi: 10.1021/bk-1993-0522.ch012

EXPERIMENTAL PROCEDURES 14

Chemicals. Radiolabeled RH-5287 (2-n-octyl- C(4,5)-dichloro-l-isothiazolin-3-one) and C RH-5287 ( C(4,5)-dichloro) were prepared at Rohm and Haas Company, Research Laboratories, Spring House, PA. The radiopurity was greater than 98% and the specific activity was 55.39 pCi/mg. The chemical purity of the C material was greater than 97%. Additional chromatographic standards were also prepared at Rohm and Haas Company. All laboratory chemicals were reagent grade and all solvents HPLC grade. 13

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Sediment and Water. The sediment and seawater used for this study were obtained from the York River near Gloucester Point, VA, and were used shortly after their collection in order to insure the viability of their natural biota. The physical properties of the sediment and seawater are listed in Tables I and II. Aerobic Microcosm. Wet sediment (55.7 g; 20 g dry weight) and 64.3 ml of seawater were added to an autoclaved 500 ml Erlenmeyer flask equipped with a ground glass stopper and stopcock inlet and outlet tubes. The inlet and outlet tubes were used to remove volatile products while providing for replacement with fresh oxygen. Sediment and seawater were not treated in any way prior to their addition to an Erlenmeyer flask. Six pg RH-5287 (1.5 pg C and 4.5 pg C) was added to each flask to yield a nominal 0.05 ppm dose. This concentration of parent compound was slightly below the minimum concentration that inhibits microbial activity and over 100 times higher than the expected environmental concentration. Dosed and controlflaskswere maintained in a dark incubator at 25 C and duplicate flasks were removed on Day 0,1, 2, 5, 9,15, 20, 26 and 30. At approximately seven day intervals oxygen wasflushedthrough theflasksand the exiting gas passed through a series of two gas dispersion tubes containing, in order, ethylene glycol and 10% NaOH. After replacing the gas dispersion tubes/solutions, the flask was brieflyflushedwith oxygen and resealed. The expired ethylene glycol solutions and the BaCl precipitated C 0 from the NaOH solutions were radioassayed. After incubation for the specified time, the seawater and sediment phases were separated by either centrifugation or filtration. The seawater phase was partitioned with dichloromethane and radioassayed. The separated sediment was extracted using a method similar to that of Rice et al. (5). The entire sediment was mixed with 22 g of Na S0 and 3 g Quso G 35 (precipitated silica, Degussa Corporation, Teterboro, NJ) and subsequently placed in a cryogenic freezer. After at least 24 hours in the freezer, the sediment mixture was transferred to a Waring blender and the resulting homogeneous mixture placed in a cellulose extraction thimble. The samples were Soxhlet extracted for 48 hours with dichloromethane:methanol (9:1) followed by a 24 hour extraction with methanol alone. 1 3

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12. JACOBSON ET AL.

Fate ofan Antifoulant in an Aquatic Environment 129

The extraction solvents were individually concentrated, radioassayed and chromatographed (HPLC). The Soxhlet-extracted sediment was stored frozen until it was exhaustively extracted. Characterization of the Soxhlet-insoluble residues followed the classical methods suggested by the U.S. Environmental Protection Agency (6). The insoluble sediment was initially refluxed in 0.25 N HC1. The resulting insoluble residue was extracted overnight with 1 Ν NaOH to yield three fractions: 1) humic acid (precipitated from alkaline solution by acid), 2) fulvic acid (that part of the alkaline solution not precipitated by acid) and 3) humin (sediment material not soluble in alkali). Anaerobic Microcosm. The anaerobic samples were treated identically to the aerobic samples with the following exceptions. On theriverbottom, the upper layer of aerobic sediment was first swept away allowing for the sampling of the lower anaerobic layer. The Erlenmeyer flask contained 54.1 g of wet sediment (20 g dry weight) and 66 ml of water purged with nitrogen. Glucose was added to each flask and the flask was flushed with nitrogen and stored in an incubator (25°C) to insure anaerobic conditions. After 30 days, C RH-5287 (5.9 pg) was added to each flask, the flasks were flushed with nitrogen, and were returned to the incubator. All manipulations were carried out in a glove box flushed constantly with nitrogen. Samples were taken on Day 0,1, 5,7,29, 61 and 90. Extraction and characterization of the two phases were identical to that of the aerobic samples. 14

HPLC. A Spectra Physics or a Waters Associates HPLC system was utilized for chromatographic analysis. Separation of compounds was accomplished with a water/methanol gradient System 1 employed a Supelco LC-18 column and the following linear gradient steps: a) 75% methanol from 0 to 15 minutes, b) 75 to 100% methanol over the next 5 minutes, c) 100% methanol for the next 15 minutes and d) 100 to 75% methanol over the next 5 minutes. System 2 employed a Supelco LC-18DB column and the following linear gradient steps: a) 5% methanol from 0 to 5 minutes, b) 5% to 95% methanol over the next 30 minutes, c) 95% methanol for the next 5 minutes and d) 95% to 5% methanol over the next 10 minutes. Flow rate for both systems was 1 ml/min and detection was with a radioactive flow monitor and a UV/visible (220 or 270 nm) detector. RESULTS AND DISCUSSION 14

Distribution of Radioactivity. The applied C-activity partitioned rapidly and primarily into the sediment. After centrifugation or filtration to separate the two phases, over 92% of the applied activity was present in the sediment throughout the study (Figures 1 and 2). In the aerobic sediment, the Soxhlet-extractableresidues(dichloromethane:methanol and methanol extractions combined) declined from about 48% of the applied activity to 16% over the 30 days of the experiment This decrease in extractableresidueswas most pronounced in the dichloromethane:methanol extraction, though the methanol extraction also decreased withtime,albeit, more slowly (data not presented). There was a steady increase in C 0 throughout the study and by Day 30 it comprised about 24% of the 1 4

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0

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30

Time (days) 14

Figure 1. Distribution of C-activity in the aerobic microcosm over time. Seawater and sediment phases were separated and the sediment extracted yielding a Soxhlet soluble and insoluble fraction.

p sol-

I

e r c

0

30

60

90

Time (days) 14

Figure 2. Distribution of C-activity in the anaerobic microcosm over time. Seawater and sediment phases were separated and the sediment extracted yielding a Soxhlet soluble and insoluble fraction.


12. JACOBSON ET AL.


applied dose. The Soxhlet-insoluble residue accounted for a majority of the applied activity, ranging between 57 to 77%. The recovery of applied C averaged 103.6 ± 10.8% over the 30 days. Resultsfromthe anaerobic sediment demonstrated trends similar to the aerobic sediment (Figure 2). There was a general decline in Soxhlet-extractableresiduewith increased incubation time. The fraction containing most of the C-activity was the Soxhlet insolubleresiduecomprising between 40 to 60% of the applied activity. Volatile production was much less than in the aerobic sediment with C 0 accounting for approximately 8% of the dose by Day 90. The recovery of applied C-activity averaged 88.1 ± 9.6% over the 90 days. Numerous extraction solvents (both organic and aqueous), extraction techniques (e.g., homogenization, sonication, shaking), as well as extraction times were examined to achieve the highest extractability of C residues from sediment. The double Soxhlet extraction procedure employed in this study was the most effective method; it extracted approximately 50% of the sediment-associated Cresidue.When sterile sediment was spiked with C RH-5287, approximately 90% of the dose was extracted by this method (data not presented). Thus it appears that biological degradation of RH-5287 is occurring and the dégradâtes may be tightly incorporated into the sediment. Microbial and chemical processes can occur within the soil or sediment whereby reactive compounds are generated leading to formation of covalendy bound residues (7). Studies on other isothiazolones (4) have similarly demonstrated atightassociation between C residues of isothiazolones and sediment/soil. The C-activity remaining after Soxhlet extraction was further characterized by exhaustive extraction with 0.25 N HC1 followed by 1 Ν NaOH (Table ΙΠ). Acid extraction had virtually no effect on the boundresidue.Treatment with base released a significant percentage of the bound residue with practically all the base-soluble C activity associated with the humic acid fraction. However, even with this severe base treatment, over 50% of the bound residue (14.5 to 40.7% of the applied dose) was insoluble (humin). Thus a large percentage of the C-activity is tightly associated with the sediment 14

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Half-life. The half-life of RH-5287 in an aerobic and an anaerobic microcosm was less than 1 hour (Figure 3, insert). Less than 6% of the extractable residue at time 0 was parent, and at subsequent sampling intervals no parent compound was detected. The small amount of parent detected attime0 may seem illogical but it took approximately 1 hour to prepare and freeze, and thus biologically inactivate the samples. Due to the necessary sample preparation, no sampling intervals of less than 1 hour were possible. When coupled with the rapid metabolism, a more detailed kinetic analysis was impossible. Fortification of sterile sediments with C RH-5287 yielded extraction efficiencies approaching 90%, and chromatography of these sediment extracts showed that over 95% of the Cresidueswas parent compound (data not presented). Thus in the presence of a biologically active microcosm, RH-5287 degraded extremely rapidly. In biologically active natural and synthetic seawater the half-life of RH-5287 was similar to the microcosmresults(8). The degradation of N-methyl isothiazolones in ariverdie-away and an activated sludge environment was also rapid (9). The N-S bond of isothiazolones 14

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Table L Physical-Chemical Characteristics of Aerobic and Anaerobic Sediment Parameter

Aerobic

Anaerobic

pH Texture Class Sand Silt Clay Organic Matter Cation Exchange Capacity Field Capacity Sulfur, Total

6.6 Silt Loam 20% 60% 20% 8.1% Dry 35 meq/100 g 58% 1.18%

6.6 Silt Loam 13% 65% 22% 5.3% Dry 29 meq/100 g 53% 1.02%

Table Π. Physical-Chemical Characteristics of Seawater Total Alkalinity Total Organic Carbon pH Salinity Total Suspended Solids Specific Conductance Sulfate Total Calcium Total Potassium Total Sodium

84 mg/L CaC03 3.2 mg/L 7.4 19.62 g/Kg 54 mg/L 32,100 pmohs/cm 2,513 mg/L 104 mg/L 266 mg/L 6,315 mg/L

Table ΠΙ. Exhaustive extraction of Soxhlet extracted sediment

Day

Percent Bound

Percent HQ Soluble

Percent in Humin Fraction

Percent in HumicAcid Fraction

Percent in FulvicAcid Fraction

Anaerobic 0

21.0

0.1

14.5

13.4

0.2

Aerobic 0 30

53.4 60.3

0.1 0.1

40.7 29.3

22.1 25.0

0.3 0.5



12. JACOBSON ET AL.


Aerobic Microcosm

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Anaerobic Metabolites —

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Aerobic C02 Aerobic Metabolites .---w-

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Anaerobic Volatiles Ο

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Figure 3. Cumulative production of C 0 and total Soxhlet soluble C metabolites detected by HPLC. Both the aerobic and anaerobic microcosms are presented. Insert represents the decline of parent over the first hour of the experiment. 2


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is very labile, undergoing rapid chemical cleavage in the presence of electrophiles or nucleophiles (2,3). Thus, degradation of RH-5287 is rapid, involving a biologically mediated attack on the N-S bond. The rapid half-life coupled with the extraction data comparing sterile and nonsterile sediment suggests that it is the dégradâtes of RH-5287 that are being rapidly and preferentially adsorbed to aquatic sediment. As outlined above, this adsorption is very strong. 14

Metabolite Characterization. By Day 3 0 , C 0 was a major degradation product of RH-5287, comprising almost 25% of the applied dose in the aerobic microcosm (Figure 3). This is an important observation since the only way to liberate C 0 is by isothiazoloneringcleavage and subsequent oxidation of the labeled methylene groups. In the reducing environment of the anaerobic microcosm, a smaller quantity of C 0 was produced. This environment would be conducive to the production of nucleophiles such as HS" and electrophiles such as CN~ which will rapidly cleave the N-S bond of isothiazolones (2,3). The production of C 0 and possibly other oxidized dégradâtes in the anaerobic environment is similar to that of alcohol and heterolactic fermentations which involve enzymatic catalysis. Figure 3 also illustrates a correlation between the decrease in total Soxhlet soluble HPLC detectable C metabolites and an increase in C 0 . This is very evident for the aerobic microcosm where by Day 30 more C 0 was present than Soxhlet-extractable C metabolites. HPLC analysis of the Soxhlet extracts is presented in Figures 4 and 5. For both aerobic and anaerobic environments, three major metabolites with retention times of approximately 3.5,4 and 7 minutes (HPLC system 1) were detected. These metabolites were chromatographically more polar than parent compound. The correlated decrease in HPLC detectable polar metabolites and the increase in C 0 over the 30 day study suggests that degradation involves successive oxidation of the ring cleaved isothiazolone (Figure 3). Co-chromatography with standards characterized these metabolites as n-octyl oxamic acid, n-octyl malonamic acid, and either n-octyl-hydroxylacetamide, n-octylglyoxylamide or the n-octyl-acetamide (Table IV). These are similar to the metabolites identified by Krezmenski et al. (9) for N-methyl isothiazolones in sludge effluent and river water. In addition, a series of isothiazolone compounds were demonstrated to undergo photoisomerization by cleavage of N-S bond (10). 2

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CONCLUSIONS When exposed to a biologically active aerobic or anaerobic microcosm consisting of seawater and sediment or seawater alone (8) RH-5287 rapidly degrades byringopening and subsequent oxidation. A major portion of these dégradâtes are tightly and preferentially associated with sediment. From these data it appears that when RH-5287 enters the environment as theresultof leaching from marine antifoulant paints it will be rapidly degraded and the bioavailability of these dégradâtes will be severely restricted by the rapid and covalent-like association with marine sediment



JACOBSON ET A L


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Time (min)

Figure 4. Chromatogram (HPLC system 1) of the Soxhlet soluble residue from the aerobic microcosm.



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PESTICIDES

IN URBAN ENVIRONMENTS

R e I a t i ν e R a d i ο a c t i ν i t

y

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Figure 5. Chromatogram (HPLC system 1) of the Soxhlet soluble residue from the anaerobic microcosm.


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Fate ofan Antifoulant in an Aquatic Environment 137

Table IV. Chromatographic Analysis of Standards in HPLC System 1

1


Name

Structure

Rt

RH-5287

23.5 C

α

S/

xs C.H

t

RH-893

10.7

Ce 17 H

N-(n-octyl)-malonamic acid

NH-C(0)-CH C0 H

3.6

N-(n-octyl)-oxamic acid

R-NH-C(0)-C0 H

4.1

N-(n-octyl)-1 -hydroxylacetamide

R-NH-C(0)-CH OH

7.0

N-(n-octyl)-glyoxylamide

R-NH-C(0)-CHO

6.8

N-(n-octyl) acetamide

R-NH-C(0)-Œ

7.3

N-(n-octyl) chloroacetamide

R-NH-C(0)-CH C1

N-(n-octyl)-chloropiOpionamide

R-NH-C(0)-CH CH Cl

2

2

2

2

3

8.3

2

r

2

1 RisC H . 8

n


9.5

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ACKNOWLEDGMENTS We gratefully acknowledge the assistance of Dr. Robert J. Huggett and his colleagues at Virginia Institute of Marine Science for providing the sediment and seawater and Dr. SouJen Chang, Rohm and Haas Company for preparation of the chromatographic standards.


LITERATURE CITED 1. 2. 3. 4. 5. 6. 7.

8.

9. 10.

Miller, G.A. and Lovegrove, T., J. Coatings Technol. 1980, 2, 69. Crow, W.D. and Nelson, N.J., J. Org. Chem. 1965, 30, 2060. Crow, W.D. and Gosney, I., Aust. J. Chem. 1967, 20, 2729. Krzeminski, S.F., Brackett, C.K. and Fisher, J.D., J. Agric. Food Chem. 1975, 23, 1060. Rice, C.D., Espourteille, F.A. and Huggett, R.J., App. Organometallic Chem. 1987, 1, 541. U.S. Environmental Protection Agency, Federal Register June 1975, 40[123], 26803. Führ. F. In Pesticide Science and Biotechnology; Greenhalgh, R and Roberts, T.R., Eds.; Sixth International Congress of Pesticide Chemistry (IUPAC); Blackwell Scientific Publication: Oxford, U.K., 1987, pp 381-89. Shade, W.D., Hurt, S.S., Jacobson, A.H., and Reinert, K.H., In Environmental Toxicology and Risk Assessment; 2nd Volume ASTM STP 1173; Gorsuch, J.W., Dwyer, F.W., Ingersoll, C.M. and La Point, T.W., Eds; American Society for Testing and Materials, Philadelphia, PA (in press). Krzeminski, S.F., Brackett, C.K., Fisher, J.D. and Spinnler, J.F., J. Agric. Food Chem. 1975, 20, 1068. Rokach, J. and Hamel, P., J.C.S. Chem. Comm. 1979, pp 786.

RECEIVED October 29,

1992


Pesticides in Urban Environments - ACS Publications - American

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