BATCH: jf2b33 USER: dle69 DIV: @xyv04/data1/CLS_pj/GRP_jf/JOB_i05/DIV_jf0257289 DATE: January 6, 2003

1 Biotransformation of an Organochlorine Insecticide,

2 Endosulfan, by Anabaena Species
3 SUNG-EUN LEE,† JONG-SOO KIM,† IVAN R. KENNEDY,‡ JONG-WOO PARK,†
4 GI-SEOK KWON,§ SUNG-CHEOL KOH,# AND JANG-EOK KIM*,†
5 Department of Agricultural Chemistry, Kyungpook National University, Daegu 702-701, Korea;
6 Department of Agricultural Chemistry and Soil Sciences, University of Sydney, NSW 2006, Australia;
7 School of Bioresource Science, Andong National University, Andong 760-749, Korea; and Department
8 of Environmenal Engineering, Korea Maritime University, Pusan 606-791, Korea

9
10 This study assesses the role of the blue-green algal species present in the soil in the dissipation of
11 endosulfan and its metabolites in the soil environment. Two Anabaena species, Anabaena sp. PCC
12 7120 and Anabaena flos-aquae, were used in this study. Anabaena sp. PCC 7120 produced three
13 principal biotransformation compounds, chiefly endosulfan diol (endodiol), and minor amounts of
14 endosulfan hydroxyether and endosulfan lactone. Trace amounts of endosulfan sulfate were detected.
15 In comparison, the biotransformation of endosulfan by Anabaena flos-aquae yielded mainly endodiol
16 with minor amounts of endosulfan sulfate. An unknown compound was produced up to 70% from
17 endosulfan spiked in the medium inoculated by A. flos-aquae after 8 days of incubation. Therefore,
18 the endosulfan fate was dependent on the species. Within 1 day of incubation, two Anabaena species
19 produced low amounts of β-endosulfan after application of R-endosulfan. These results suggest the
20 presence of isomerase in the Anabaena species. Further studies using a fermentor to control the
21 medium pH at 7.4 to minimize chemical hydrolysis of endosulfan revealed a major production of
22 endodiol with minor amounts of endosulfan sulfate and the unknown compound. These results showed
23 that the production of the unknown compound might be dependent on the alkaline pH in the medium
24 and that the production of endodiol by A. flos-aquae might be biologically controlled. This study showed
25 that two algal species could contribute in the detoxification pathways of endosulfan in the soil
26 environment.
27

28 KEYWORDS: Endosulfan; endosulfan diol; endosulfan sulfate; Anabaena flos-aquae; Anabaena sp. PCC
29 7120; fermentor

30 INTRODUCTION (endodiol) are known to be produced by oxidation and hydroly- 44

sis, respectively. Endodiol is a nontoxic metabolite to fish and 45
31 Even though significant increases in agricultural productivity
other organisms; thus, hydrolysis producing endodiol may be 46
32 have resulted from the control of agricultural pests with synthetic
33 chemical pesticides (1), there is widespread concern about the an important detoxification pathway of endosulfan. However, 47

34 presence of pesticide residues in food and the environment. endosulfan sulfate has a similar toxicity compared to the parent 48

35 Endosulfan (1,2,5,6,7,7-hexachloro-5-norbornene-2,3-dimetha- compound endosulfan. In addition, endosulfan sulfate has a 49

36 nol cyclic sulfite) is an organochlorine insecticide mainly used much longer tolerance in the soil environment in comparison 50

37 to control HelicoVerpa species in the upland soil in Korea. Fish to endosulfan (11). Therefore, the production of endosulfan 51

38 are very susceptible to endosulfan toxicity at a level of 1-20 sulfate seems to cause long persistence of endosulfan in soil. 52

39 ng/L. Several intensive studies on the degradation of endosulfan Endosulfan sulfate is produced by several microorganisms 53

40 in soil or water environments have been conducted (2-10). including Phanerochaete chrysosporium (2, 3), Mucor thermo- 54

41 There are two principal mechanisms of endosulfan degradation hyalospora MTCC 1384 (4), and Trichoderma harzianum (5). 55

42 due to the oxidation or hydrolysis caused by chemical or However, the last two fungi produce endodiol as a major 56

43 biological systems. Endosulfan sulfate and endosulfan diol endosulfan metabolite in culture medium. Martens (6) reported 57
the degradation of endosulfan to endodiol as a primary 58

* Corresponding author (telephone 82-53-950-5720; fax 82-53-953-7233;

metabolite followed by endosulfan sulfate in flooded soil, when 59
e-mail jekim@knu.ac.kr). incubated with several fungi species. Guerin and Kennedy (7) 60
† Kyungpook National University.
‡ University of Sydney.
and Guerin (8) detected the formation of endodiol when 61
§ Andong National University. endosulfan was incubated with bacteria under anaerobic condi- 62
# Korea Maritime University. tions. In addition, Miles and Moy (9) studied extensive 63

10.1021/jf0257289 CCC: $25.00 © xxxx American Chemical Society

Published on Web 00/00/0000 PAGE EST: 4.4
BATCH: jf2b33 USER: dle69 DIV: @xyv04/data1/CLS_pj/GRP_jf/JOB_i05/DIV_jf0257289 DATE: January 6, 2003

B Lee et al.

64 degradation of endosulfan in an aqueous nutrient medium

65 inoculated with mixed soil microorganisms and identified the
66 degradation pathway of endosulfan including the formation of
67 different metabolites such as endosulfan sulfate, endodiol,
68 endosulfan ether, endosulfan hydroxyether, and endosulfan
69 lactone. Recently, Sutherland et al. (10) have found a tentative
70 metabolite endosulfan monoaldehyde as a hydrolysis metabolite
71 and endosulfan sulfate in a strongly buffered culture medium
72 (pH 6.6) to give a minimum chemical hydrolysis.
73 Cyanobacteria are free living, photoautotrophic microorgan-
74 isms that have shown their capabilities to degrade both naturally
75 occurring compounds and synthetic chemicals, especially pes-
76 ticides (12-14). Therefore, cyanobacteria have been considered
77 to be potent alternative organisms for chemical and physical Figure 1. Growth of A. flos-aquae and change of the medium pH.
78 treatments to transform environmentally persistent, toxic materi-
79 als. For example, three blue-green algae, Synechococcus elon-
80 gates, Nostoc linckia, and Phormidium tenue, strongly participate
81 in the degradation of monocrotophos and quinalphos in soil (14).
82 Here, we report the biotransformation of endosulfan by two blue-
83 green algal species, Anabaena sp. PCC 7120 and Anabaena flos-
84 aquae, in a culture medium and the metabolites. We also
85 postulate the role of the two Anabaena species in the soil
86 environment to dissipate endosulfan.

87 EXPERIMENTAL PROCEDURES
88 Microorganisms. Anabaena sp. PCC 7120 and A. flos-aquae were
89 obtained from American Type Culture Collection (ATCC) and Korea
90 Research Institute of Bioscience and Biotechnology, respectively.
91 Anabaena species were grown in Allen’s liquid medium without nitrate
92 on a shaker at room temperature in a light intensity of ∼1700 lx by
93 fluorescent lamps with a 12 h/12 h (light/dark) cycle. Their growth
94 was monitored by measurement of chlorophyll a content (15). All
95 operations were carried out under sterile conditions in order to avoid
96 bacterial contamination.
97 Chemicals. R-Endosulfan, β-endosulfan, endosulfan sulfate, en-
98 dodiol, endosulfan ether, and endosulfan hydroxyether were purchased
99 from Chem Service Inc. (West Chester, PA). NaNO3, K2HPO4, MgSO4‚
100 7H2O, CaCl2, ferric citrate, and ethylenediaminetetraacetate (EDTA)
101 were purchased from Sigma Chemical Co. (St. Louis, MO). All
102 chemicals used were of the highest grade commercially available.
103 Determination of Endosulfan Degradation and Metabolite Pro-
104 duction. Experiments were carried out in batch cultures. One hundred
105 milliliters of Allen’s medium in 250 flasks stoppered with cotton plugs
106 was inoculated with each Anabaena species. Four days later, R-en-
107 dosulfan was supplemented to the inoculated medium to give a final
108 concentration of 10 µg/mL. An uninoculated culture medium served
109 as a control. All of the flasks were sealed and incubated on a shaker at
110 room temperature in a light intensity of ∼1700 lx by fluorescent lamps
111 with a 12 h/12 h (light/dark) cycle. Algal growth was monitored by Figure 2. Endosulfan degradation after spiking in the medium inoculated
112 measuring chlorophyll a content (15). Degradation was assessed by with A. flos-aquae: (a) endosulfan remaining (%); (b) production of
113 measurement of endosulfan in triplicate flasks. Sampling was done at endodiol; (c) production of unknown compound.
114 various periods of incubation and analyzed by gas chromatography with
115 an electron capture detector (GC-ECD). For a pH-controlled experiment, RESULTS AND DISCUSSION 131
116 all procedures were conducted the same as above. However, 1 L of
117 Allen’s medium in a Bioneer fermentor (Bioneer Co., Seoul, Korea) The growth of A. flos-aquae continued for up to 15 days after 132
118 was inoculated by A. flos-aquae. Sampling (10 mL) was done at various inoculation, and then the growth ceased (Figure 1). The increase 133
119 periods of incubation. The pH value of the fermentor was set to 7.2. of pH in the medium was found until 10 days after inoculation 134
120 The remaining endosulfan and its metabolites were extracted from with A. flos-aquae. Chlorophyll a content and pH in the A. flos- 135
121 5 mL of the crushed bacterial suspension by a glass homogenizer with aquae inoculated medium was up to about 50 mg/L and 12.5, 136
122 an equal volume of nanograde hexane by vortexing for 30 s twice. respectively. Endosulfan spiked after 7 days of inoculation. 137
123 The organic layer was collected in a vial and dried with nitrogen gas. However, there were no changes in either chlorophyll a content 138
124 Two milliliters of hexane was added to the dried sample, and a 2 µL
or pH in the control medium (Figure 1). Endosulfan concentra- 139
125 volume of each hexane extract was subjected to GC-ECD analysis in
126 a Varian Star 3400 CX with an electron capture detector on a DE-5 tion decreased immediately after spiking into the medium, 140

127 fused silica capillary column (30 m × 0.32 mm i.d. with 0.25-mm declining after 4 days of incubation to <10%, as shown in 141
128 film coating) in a linear temperature gradient from 110 to 190 °C over Figure 2a. Endosulfan spiked in the control was not dissipated 142
129 8.5 min. Chromatographic patterns were analyzed with Turbochem during this time. The major metabolite detected was endodiol 143
130 software (Seoul, Korea). (Figure 2b), and endosulfan sulfate and β-endosulfan were also 144
BATCH: jf2b33 USER: dle69 DIV: @xyv04/data1/CLS_pj/GRP_jf/JOB_i05/DIV_jf0257289 DATE: January 6, 2003

Biotransformation of Endosulfan by Anabaena Species C

parathion-methyl to 4-nitrophenol in the medium. Anabaena sp. 164

PCC 7120 possesses dechlorination activity of lindane (17, 18), 165
leading to the formation of 2,3,4,5,6-pentachloro-1-cyclohexene 166
and 1,2,3- and 1,2,4-trichlorobenzene. Anabaena sp. can trans- 167
form 2,4,6-trinitrotoluene (TNT) to azoxytetranitrotoluene and 168
hydroxyaminodinitrotoluene (19). However, several studies have 169
shown some impact of pesticides on the growth of cyanobac- 170
teria. Mohapatra and Mohanty (20) demonstrated that dimethoate 171
and endosulfan inhibited growth and decreased survivability of 172
Anabaena doliolum. Atrazine and hexazinone also inhibited the 173
growth of Anabaena flos-aquae and Selenastrum capricornutum 174
(21). Therefore, the impact of pesticides on the growth of 175
Anabaena species can reduce the removal or dissipation of 176
pesticides in soil. 177
Our results show evidence of two metabolic pathways of 178
endosulfan by two different Anabaena species. Two Anabaena 179
species produced endodiol as a primary product and a trace 180
amount of endosulfan sulfate. Endodiol is a nontoxic compound; 181
thus, we believe this is a hydrolysis pathway for a detoxification 182
of endosulfan in the soil environment. However, the question 183
arises how they produced endodiol. As we showed, because 184
there is an increase of pH in the medium, chemical hydrolysis 185
might influence the rate of endodiol production. None of the 186
enzymes has been reported for endosulfan hydrolysis. One 187
interesting study has been done investigating the biological 188
hydrolysis of endosulfan in pure culture medium (10). The 189
authors suggested the use of a strongly buffered culture medium 190
(pH 6.6) to minimize chemical hydrolysis of endosulfan. In 191
addition, the medium included the detergent Tween 80 for 192
increasing the amount of endosulfan in contact with mixed 193
bacteria. No bacterial growth was detected in the control cultures 194
in the absence of endosulfan as a carbon source. 195
In the presence of endosulfan, growth of a mixed culture of 196
bacteria occurred concomitantly with endosulfan decrease (10). 197
Endosulfan was subjected to degradation by oxidation and 198
hydrolysis. Conclusively, endosulfan sulfate formation was 199
found to be favored as oxidative production, and a novel 200
hydrolysis product tentatively identified as endosulfan monoal- 201
dehyde was found. From these findings, the two factors of pH 202
and contact seem to be very important for enzymic endosulfan 203
degradation by microorganisms. There was one more distinct 204
Figure 3. Endosulfan degradation after spiking in the medium inoculated
factor remaining as oxygen because the biological oxidation 205
with Anabaena sp. PCC 7120: (a) algal growth in the medium; (b)
reaction needs a supply of oxygen. 206
degradation of endosulfan spiked and the production of endodiol; (c)
production of metabolites. Therefore, we assume that the endodiol production in our 207
study might result from chemical hydrolysis due to the increase 208
145 found in trace amounts. The endodiol produced disappeared of pH in the medium alone. However, we could not exclude 209
146 gradually, with 20% of the initial endosulfan remaining as the participation of biological hydrolysis because the production 210
147 endodiol after 8 days from spiking. Interestingly, an unknown of endosulfan sulfate and endosulfan hydroxyether, as shown 211
148 compound was detected that increased with incubation time as in Figures 2 and 3, could involve biological oxidation in both 212
149 the basis of the detected peak area in GC chromatogram as Anabaena species. To understand the mechanism of production 213
150 shown in Figure 3c. However, there is no indication of what of endodiol in the medium, we used a fermentor to prevent the 214
151 proportion of the total endosulfan became the unknown peak. increase of the medium pH and to supply oxygen in the medium 215
152 On the other hand, Anabaena sp. PCC 7120 produced endodiol constantly to protect against oxygen shortages. These conditions 216
153 and endosulfan hydroxyether as the principal metabolites and may enhance the biological oxidation of endosulfan in the 217
154 endosulfan sulfate and β-endosulfan as minor metabolites medium. 218
155 (Figure 3b,c). Anabaena sp. PCC7120 also produced the A. flos-aquae in the pH-controlled experiment using a 219
156 unknown compound. However, the amount of the compound fermentor produced mainly endodiol (Figure 4). Chlorophyll 220
157 produced was insignificant. a content was increased in the A. flos-aquae inoculated medium 221
158 Cyanobacteria can degrade both naturally occurring aromatic and was used to monitor growth, but no growth occurred in the 222
159 hydrocarbons and man-made xenobiotics. Cerniglia et al. (16) control (Figure 4a). The spiked endosulfan dramatically 223
160 tested the Oscillatoria sp. strain JCM for naphthalene metabo- declined after 2 days of incubation, with 20% of the endosulfan 224
161 lism and showed formation of 1-naphthol from naphthalene. remaining after 7 days of incubation. After 4 days of incubation, 225
162 Four species of cyanobacteria, such as N. linkia, N. muscorum, the endosulfan spike gradually disappeared and reached 50% 226
163 Oscillatoria animalis, and Phormidium foVeolarum, can degrade in control medium (Figure 4b). Endodiol was produced and 227
BATCH: jf2b33 USER: dle69 DIV: @xyv04/data1/CLS_pj/GRP_jf/JOB_i05/DIV_jf0257289 DATE: January 6, 2003

D Lee et al.

Scheme 1 Proposed Endosulfan Biotransformation Pathway by

Anabaena Species

Figure 4. Endosulfan degradation after spiking in a medium inoculated

with A. flos-aquae using a fermentor controlling the pH at 7.2: (a) algal
growth in the medium; (b) endosulfan remaining (%) and the production
of endodiol (%).

228 endosulfan sulfate was detected in trace amounts. In this

229 experiment, only a small amount of the unknown compound
230 was determined, <1%, as the basis of the peak area expressed
231 in the GC chromatogram. Even when the pH in the medium salt ions present in agricultural soils on endosulfan biotrans- 260
formation by Anabaena species. 261
232 was controlled, A. flos-aquae produced abundant endodiol. With
233 these findings, there may be at least two mechanisms to produce
LITERATURE CITED 262
234 endodiol.
235 There are several more considerable factors for microorgan- (1) Duke, S. O.; Menn, J. J.; Plimer, J. R. Challenges of pest control 263
236 isms living in soil. Photolysis may be another factor. Therefore, with enhanced toxicological and environmental safety. In ACS 264
237 endodiol production can be significantly influenced by pH and Symposium Series 524; Duke, S. O., Menn, J. J., Plimer, J. R., 265
238 light. As many studies have suggested, cyanobacteria including Eds.; American Chemical Society: Washington DC, 1993; pp 266
1-13. 267
239 Anabaena species are present in soil. In agricultural soils, high
(2) Kullman, S. W.; Matsumura, F. Metabolic pathways utilized by 268
240 water potential is determined as the presence of salt ions. This Phanerochaete chrysosporium for degradation of the cyclodiene 269
241 strong water potential may contribute to or enhance biotrans- pesticide endosulfan. Appl. EnViron. Microbiol. 1996, 62, 593- 270
242 formation of pesticides by soil microorganisms. Han and New 600. 271
243 (22) suggested maximal removal of 2,4-dichlorophenoxyacetic (3) Kim, Y.-K.; Kim, S.-H.; Choi, S.-C. Kinetics of endosulfan 272
244 acid (2,4-D) by soil organisms occurring at the highest water degradation by Phanerochaete chrysosporium. Biotechnol. Lett. 273
245 potential (ψ) of -0.1 MPa, and degradation decreased progres- 2001, 23, 163-166. 274
246 sively down to ψ ) -5.5 MPa with no breakdown at ψ ) -22 (4) Shetty, P. K.; Mitra, J.; Murthy, N. B. K.; Namitha, K. K.; Savita, 275

247 MPa. Awasthi et al. (23) also identified moisture content as K. N.; Raghu, K. Biodegradation of cyclodiene insecticide 276
endosulfan by Mucor thermo-hyalospora MTCC 1384. Curr. Sci. 277
248 one of the most influential factors in endosulfan degradation.
2000, 79, 1381-1383. 278
249 They demonstrated pH, concentration of endosulfan, and size (5) Katayama, A.; Matsumura, F. Degradation of organochlorine 279
250 of inoculum to be the principal factors in endosulfan degrada- pesticides, particularly endosulfan, by Trichoderma harzianum. 280
251 tion. EnViron. Toxicol. Chem. 1993, 12, 1059-1065. 281
252 In conclusion, in two different experiments we have shown (6) Martens, R. Degradation of [8,9-14C]endosulfan by soil micro- 282
253 that endosulfan transformation by two Anabaena species occurs organisms. Appl. EnViron. Microbiol. 1977, 31, 853-858. 283

254 by oxidation and hydrolysis reactions. In addition, both Ana- (7) Guerin, T. F.; Kennedy, I. R. Distribution and dissipation of 284
endosulfan and related cyclodienes in sterile aqueous systems: 285
255 baena species participate in the detoxification process of implications for studies on biodegradation. J. Agric. Food Chem. 286
256 endosulfan in soil to produce endodiol as a nontoxic metabolite. 1992, 40, 2315-2323. 287
257 We have proposed a possible biotranformation pathway of (8) Guerin, T. F. The anaerobic degradation of endosulfan by 288
258 endosulfan by Anabaena species, as shown in Scheme 1. Further indigenous microorganisms from low-oxygen soils and sedi- 289
259 studies are needed to evaluate the impact of water potential by ments. EnViron. Pollut. 1999, 106, 13-21. 290
BATCH: jf2b33 USER: dle69 DIV: @xyv04/data1/CLS_pj/GRP_jf/JOB_i05/DIV_jf0257289 DATE: January 6, 2003

Biotransformation of Endosulfan by Anabaena Species PAGE EST: 4.4 E

291 (9) Miles, J. R. W.; Moy, P. Degradation of endosulfan and its (18) Kuritz, T.; Bocanera, L. V.; Riveria, N. S. Dechlorination of 321
292 metabolites by a mixed culture of soil microorganisms. Bull. lindane by the cyanobacterium Anabaena sp. strain PCC7120 322
293 EnViron. Contam. Toxicol. 1979, 23, 13-19. depends on the function of the nir operon. J. Bacteriol. 1997, 323
294 (10) Sutherland, T. D.; Horne, I.; Lacey, M. J.; Harcourt, R. L.; 179, 3368-3370. 324
295 Russell, R. J.; Oakeshott, J. G. Enrichment of an endosulfan- (19) Pavlostathis, S. G.; Jackson, G. H. Biotranformion of 2,4,6- 325
296 degrading mixed bacterial culture. Appl. EnViron. Microbiol. trinitrotoluene in Anabaena sp. cultures. EnViron. Toxicol. Chem. 326
297 2000, 66, 2822-2828. 1999, 18, 412-419. 327
298 (11) Kennedy, I. R.; Sanchez-Bayo, F.; Kimber, S. W.; Hugo, L.;
(20) Mohapatra, P. K.; Mohanty, R. C. Growth pattern changes of 328
299 Ahmad, N. Off-site movement of endosulfan from irrigated
Chlorella Vulgaris and Anabaena dolilum due to toxicity of 329
300 cotton in New South Wales. J. EnViron. Qual. 2001, 30, 683-
301 696. dimethoate and endosulfan. Bull. EnViron. Contam. Toxicol. 330
302 (12) Yan, G. A.; Jiang, J. W.; Wu, G.; Yan, X. Disappearance of 1992, 49, 576-581. 331
303 linear alkylbenzene sulfinate from different cultures with Ana- (21) Abou-Waly, H.; Abou-Setta, M. M.; Nigg, H. N.; Mallory, L. 332
304 baena sp. HB 1017. Bull. EnViron. Contam. Toxicol. 1998, 60, L. Growth response of freshwas algae, Anabaena flos-aquae and 333
305 329-334. Selenastrum capricornutum to atrazine and hexazinone herbicide. 334
306 (13) Megharaj, M.; Madhavi, D. R.; Sreenivasulu, C.; Umamaheswari, Bull. EnViron. Contam. Toxicol. 1991, 46, 223-229. 335
307 A.; Venkateswarlu, K. Biodegradation of methyl parathion by (22) Han, S. O.; New, P. B. Effect of water availability on degradation 336
308 soil isolates of microalgae and cyanobacteria. Bull. EnViron. of 2,4-dichlophenoxyacetic acid (2,4-D) by soil microorganism 337
309 Contam. Toxicol. 1994, 53, 292-297. Soil Biol. Biochem. 1994, 26, 1689-1697. 338
310 (14) Megharaj, M.; Venkateswarlu, K.; Rao, A. S. Metabolism of (23) Awasthi, N.; Ahuj, R.; Kumar, A. Factors influencing the 339
311 monocrotophos and quinalphos by algae isolated from soil. Bull. degradation of soil-applied endosulfan isomers. Soil Biol. Bio- 340
312 EnViron. Contam. Toxicol. 1987, 39, 251-256. chem. 2000, 32, 1697-1705. 341
313 (15) Mackinney, G. Absorption of light by chlorophyll solutions. J.
314 Biol. Chem. 1941, 140, 315-322. 342
315 (16) Cerniglia, C. E.; Van Baalen, C.; Gibson, D. T. Metabolism of Received for review June 7, 2002. Revised manuscript received 343
316 naphthalene by the cyanobacterium Oscillatoria sp., strain JCM. November 13, 2002. Accepted December 5, 2002. This study was 344
317 J. Gen. Microbiol. 1980, 116, 485-494. supported by Grant RO1-2000-000-00196-0 from the Basic Research 345
318 (17) Kuritz, T.; Wolk, C. P. Use of filamentous cyanobacteria for Program of the Korea Science and Engineering Foundation. 346
319 biogdegradation of organic pollutants. Appl. EnViron. Microbiol.
320 1995, 61, 234-238. JF0257289 347