Biological Methods for the Disposal of Coumaphos Waste - American

Chapter 19

Biological Methods for the Disposal of Coumaphos Waste Downloaded by UNIV MASSACHUSETTS AMHERST on August 2, 2012 | http://pubs.acs.org Publication Date: October 30, 1992 | doi: 10.1021/bk-1992-0510.ch019

D. R. Shelton, Jeffrey S. Karns, and Cathleen J. Hapeman-Somich Pesticide Degradation Laboratory,BeltsvilleAgricultural Research Center, Agricultural Research Service, U.S. Depeartment of Agriculture, Beltsville, MD 20705

Two methods have been developed for the disposal of large volumes of spent coumaphos, a livestock tick acaricide, generated annually from cattle dipping operations. The first involves hydrolysis of coumaphos using an organophosphate hydrolase enzyme, followed by ozonation of the hydrolysis products. The resulting ozonation products are readily mineralized by soil microorganisms. The second method involves coumaphos mineralization by a microbial consortium indigenous to one of the vats. The metabolic pathway was partially elucidated and the most important consortium members isolated and characterized. Optimal fermentation parameters were defined. Both methods have been successfully demonstrated at the bench-scale.

Coumaphos [Ο,Ο-diethyl 0-(3-chloro-4-methyl-2-oxo-2//-l-benzopyran-7-yl phosphorothioate] is used as an acaricide for control of the southern cattle tick (Boophilus microplus) and the cattle tick (Boophilus annulatus) by the Animal and Plant Health Inspection Service (APHIS), U.S. Department of Agriculture (USDA), in its Tick Eradication Program. Several hundred thousand head of cattle are dipped annually using approximately 60 vats located along the U.S.-Mexican border. The vats contain approximately 15,000 L of coumaphos solution, 0.15-0.30 % active ingredient, in the form of Co-Ral Wettable Powder or Co-Ral Flowable Liquid formulations (1). Vats are emptied, cleaned, and recharged annually, or more frequently depending on the number of cattle treated, sediment accumulation, or loss of efficacy. Cumulatively, approximately 10 L of coumaphos waste is generated annually, and is disposed of by pumping into evaporation pits present at each vat-site. 6

This chapter not subject to U.S. copyright Published 1992 American Chemical Society In Pesticide Waste Management; Bourke, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Downloaded by UNIV MASSACHUSETTS AMHERST on August 2, 2012 | http://pubs.acs.org Publication Date: October 30, 1992 | doi: 10.1021/bk-1992-0510.ch019

19. SHELTON ET AL.

Biological Methods for Disposal of Coumaphos Waste 217

Several strategies were considered for more environmently sound disposal of coumaphos waste. Early experiments had indicated that a Flavobacterium sp. (2), isolated for its ability to hydrolyze organophosphorous insecticides (3), could also hydrolyze coumaphos to chlorferon (3-chloro-4-methyl-7hydroxycoumarin). This product was readily mineralized via a combination of UV-ozonation and microbiological treatment. In a field trial, 600 L of cattle dip solution was successfully treated by growing the Flavobacterium sp. in the solution amended with xylose as growth substrate, ammonium sulfate, and phosphate (4). In a subsequent field trial, however, the Flavobacterium sp. failed to grow, apparently due to a deficiency in metal ions (5), which suggested that the growth of Flavobacterium sp. directly in cattle dip solutions was not practical. Research was initiated to develop a more reliable method for coumaphos disposal using a combination of enzymatic hydrolysis and ozonation techniques (the enzymatic/ozonation approach). A second strategy arose from studies to determine the reason(s) for a loss of efficacy in the field, i.e., accelerated rates of coumaphos degradation in several high use vats. These experiments revealed that coumaphos was reductively dechlorinated to potasan [0,0-diethyl 0-(4-methyl-2-oxo-2tf-lbenzopyran-7-yl) phosphorothioate] under anaerobic conditions and that both potasan and coumaphos were subsequently mineralized under aerobic conditions (6). Research was undertaken to isolate the responsible microorganisms and to elucidate the pathways of coumaphos metabolism, and to determine if these microorganisms indigenous to the vats could be exploited for the disposal of the waste coumaphos solutions (the microbial fermentation approach). Enzymatic/Ozonation Approach Studies were conducted with cell-free enzyme extracts to optimize the conditions for coumaphos hydrolysis. Enzyme extracts were derived from cultures of Flavobacterium sp., or recombinant strains of Esherichia coli (7) or Streptomyces lividans (8) containing the cloned organophosphate hydrolase gene. Due to the very low solubility of coumaphos (50 ppb), experiments were undertaken to assess the effect of a non-ionic detergent (Tween 80) on the rate of hydrolysis. Increasing concentrations of detergent had a dramatic effect on the rate of coumaphos hydrolysis (Table I). Since previous research had indicated that cobalt or copper were stimulatory to rates of organophosphate hydrolase activity with parathion as the substrate (5), experiments were conducted to assess the effect of cobalt sulfate on rates of coumaphos hydrolysis. Increasing concentrations of cobalt also increased the rate of coumaphos hydrolysis (Table I). TABLE I. Rate of Coumaphos Hydrolysis bv Organophosphate Hydrolase Percent First Order Rate Constant Concentration Tween Cobalt 0 0.0048 0.0019 0.001 0.0065 0.0019 0.010 0.0140 0.0023 0.100 0.0220 0.0042

In Pesticide Waste Management; Bourke, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


218

PESTICIDE WASTE MANAGEMENT

A bench-scale experiment with 76 L of dip vat solution was undertaken to demonstrate the feasibility of the enzymatic/ozonation approach. The pH of the waste solution was adjusted to between 9.5 and 10.0, and 0.05% (v/v) of Triton X-100 was added. Twenty milliliters of a crude enzyme extract (from Flavobacterium sp.) containing 19,760 International Units of parathion hydrolase activity was added at time zero. Hydrolysis of coumaphos to chlorferon and potasan to 4-methylumbelliferone was complete within 48 hours (Figure 1). Subsequent ozonation of chlorferon and 4methylumbelliferone resulted in their complete transformation to numerous unidentified products within 10 hours (Figure 2). Previous research had demonstrated that ozonation products are readily mineralized by soil microorganisms (2). Microbial Fermentation Approach Microbial enrichment cultures were started from samples of cattle dip solution using the flowable liquid formulation (42% coumaphos, 58% inert ingredients) as a sole energy and carbon source. After several transfers (10% inoculum), attempts were made to isolate the strains responsible for coumaphos metabolism. Three bacterial strains capable of coumaphos hydrolysis were obtained, but only one strain (B-l) was capable of further metabolism of chlorferon. This strain, however, was not able to utilize

3.0 2.5 Λ

-0.5 10

20

30

40

HOURS

Figure 1. Hydrolysis of coumaphos and potasan in 76 L of cattle-dip waste with organophosphate hydrolase (260 I.U./L) from Flavobacterium sp. (•) coumaphos; (Δ) chlorferon; (#) potasan; (O) methylumbelliferone.



19. SHELTONETAL.

Biological Methods for Disposal of Coumaphos Waste 219

chlorferon as a growth substrate because one or more chlorferon metabolites were inhibitory to growth. B-l was able to utilize 4-methylumbelliferone as a growth substrate with no apparent inhibitory effects. Another strain (B-4) was subsequently isolated which, when combined with B-l, resulted in the utilization of coumaphos and chlorferon as a growth substrate (Figure 3). Presumably, B-4 was able to utilize the inhibitory chlorferon metabolite(s) produced by B - l as growth substrates, thereby removing them from solution and allowing the growth of both strains. Approximately 90% of the chlorferon was mineralized, with about 10% transformed to a metabolite, achloro-)9-methyl-2,5,4-trihydroxy-imAw-cirmarriic acid, (CMTC) which was degraded by one or more unidentified bacteria (Figure 4) (9). An analagous metabolite (MC) was produced from 4-methylumbelliferone by B-l. The other hydrolysis product of coumaphos and potasan, diethylthioposphoric acid (DETP), was mineralized by a different consortium of microorganisms (10). Bench-scale experiments with 1 L samples of cattle dip solutions from several vats containing either the flowable liquid or wettable powder formulation were conducted to determine if the microbial consortium could be used to dispose of cattle dip wastes. Solutions were buffered at pH 6.8-7.2 and amended with 100 ppm yeast extract as a nutritional supplement; samples were incubated for 10 to 14 days at 23°C. With the exception of San Andreas and Pinto (mechanical failures) degradation of coumaphos generally exceeded 99% and final coumaphos concentrations were less than 10 ppm (Table II) (11). Comparison of Approaches Since neither disposal technology has been attempted at the pilot-scale, precise comparisons are not possible, however, it is possible to make qualitative judgments. The enzymatic/ozonation approach is likely to be more expensive due to both higher fixed and capital costs. Because the enzyme is degraded over time, this approach requires a constant supply of enzyme. Enzyme production costs will be dependent upon the scale of production and the strain used. Organophosphate hydrolase is membrane bound in Flavobacterium sp., but soluble in the recombinant strains E. coli and Streptomyces lividans, suggesting that production costs may be lower with the recombinant strains due to the ease of enzyme recovery. Alternatively, the development of immobilized enzyme technologies may allow the reuse of the enzyme, thereby lowering costs. However, many technical difficulties remain to be overcome before this would be practical. Other fixed costs would include the operation and maintenance of the ozone generator and ozonation vessel. Capital costs would include the purchase of the ozone generator and ozonation vessel. The trade off for higher cost is greater reliability. The amount of enzyme can be readily changed depending on the coumaphos concentration or other factors effecting enzyme activity. Likewise, the length of time of ozonation can be varied so as to achieve complete destruction of chlorferon. In contrast to the enzyme/ozonation approach, the microbial fermentation approach is likely to be less expensive but also less reliable. Fixed costs are minimal since fermentation of the contents of one vat results in the production of inoculum for other vats. A source of organic and/or inorganic


220

PESTICIDE WASTE MANAGEMENT 3.0


2.0

ΔΔ

Ο 1.0

Οo o , Ο Q Or

"ΔΛ

Δ

Δ.

0.0 100

400

300

200

500

MINUTES Figure 2. Destruction of chlorferon (Δ) and methylumbelliferone (O) by ozone, previously treated with organophosphate hydrolase.

1000

1

1

I

ι

10

800

600

400 C0UMAPH0S\ /B-4 200

((•

ι

1

3

1

1

4

5

1

1

DAYS Figure 3. Utilization of coumaphos as a growth substrate by a twomembered consortium consisting of B-l and B-4. (•) coumaphos; (O) CFU, colony forming units. (Reproduced from 11 Butterworth-Heinemann.)


In Pesticide Waste Management; Bourke, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992. 4-METHYLUMBELLIFERONE

Figure 4. Partial pathway of coumaphos (B-l and B-4) and potasan (B-l) metabolism. Potasan was produced as a result of reductive dechlorination of coumaphos. DETP was mineralized by a separate bacterial consortium. CMTC and MC were metabolized by one or more unidentified microorganisms. (Reproduced from 11 Butterworth-Heinemann.)

POTASAN

P-0


ÇOOH

a

ι 1

ι

>

r

S3

ο

w

222

PESTICIDE WASTE MANAGEMENT

Table II. Coumaphos Degradation in Vat Dip bv Microbial Consortium Vat (Formulation) Laredo Import (FL) Juarez (FL) Laredo City (WP) Zapata (WP) Calaboz (WP) San Andres (WP) Pinto' (WP) 1


2

1

2 3

Number of Final Cattle Concentration fmg/L) Dipped 11,923 3 32,252 3 951 4 4 375 4 387 12 451 10

Percent Degradation 99.8 99.7 99.6 99.7 99.8 98.8 97.4

F L = Flowable Liquid, initial coumaphos concentration ca. 0.15% WP = Wettable powder, initial coumaphos concentration ca. 0.1% pH probe failed resulting in low pH. Aeration and agitation failed.

nutrients may be needed, as well as acid and base to control pH, but these amendments and reagents are comparatively inexpensive. Capital costs include a fermentation vessel with aeration system and pH control. Temperature controls are probably not needed since disposal could presumably be scheduled for spring and fall seasons when daily temperatures would be conducive to rapid rates of degradation. Despite the apparent hardiness of the culture, microbial fermentation may be less reliable than the enzyme ozonation approach. Although, in the lab, the method appears to be reliable based on the limited number of vats tested, unknown inhibitors or fluctuations in metal ion composition in some vats may render the method ineffective. At even a low frequency of occurrence, this could result in a loss of cost effectiveness since a back-up method (enzymatic/ozonation) would be required. Further pilot-scale research with both methods will be required in order to answer these questions.

Literature Cited 1. Rogers, B. Animal and Plant Health Inspection Service, USDA, personal communication, 1990. 2. Kearney, P.C.; Karns, J.S.; Muldoon, M.T.; Ruth, J.M. J. Agric. Food Chem. 1986, 34, 702-706. 3. Sethunathan, N.; Yoshida, T. Can. J. Microbiol. 1973, 19, 873-875. 4. Karns, J.S.; Muldoon, M.T.; Mulbry, W.W.; Derbyshire, K.; Kearney, P.C. In Biotechnology in Agricultural Chemistry; LeBaron, H.M.; Mumma, R.O.; Honeycutt, R.C.; Duesing, J.H., Eds.; ACS Symposium Series No. 334; American Chemical Society: Washington, DC, 1987; pp 156-170. 5. Karns, J.S. Pesticide Degradation Laboratory, USDA/ARS, unpublished data. 6. Shelton, D.R.; Karns, J.S. J. Agric. Food Chem. 1988, 36, 831-834.


19.SHELTONETAL.BiologicalMethodsforDisposalofCournaphosWaste223


7. Mulbry, W.W.; Karns, J.S. J. Bacteriol. 1989, 171, 6740-6746. 8. Steiert, J.G.; Pogell, B.M.; Speedie, M.K.; Laredo, J. Bio/Technology 1989 7, 65-68. 9. Shelton, D.R.; Hapeman-Somich, C.J. Appl. Environ. Microbiol. 1988, 54, 2566-2571. 10. Shelton, D.R. Appl. Environ. Microbiol. 1988, 54, 2572-2573. 11. Shelton, D.R.; Hapeman-Somich, C.J. In On-Site and In Situ Bioreclamation; Hinchee, R.E.; Olfenbuttel, R.F., Eds., ButterworthHeinemann: Boston, MA, 1991; pp 313-323. RECEIVED May 12, 1992


Biological Methods for the Disposal of Coumaphos Waste - American

Recommend Documents