J. appl. Bact. 1976,40, 67-72
Degradation of Microbicides under Different Environmental Conditions
J. P. VOETS, P. PIPYN, P. V A N LANCKER AND W. VERSTRAETE laboratory of General and Industrial Microbiology, State University of Gent, Coupure 533, 9000 Gent, Belgium Received 23 June 1975 and accepted 2 September 1975 To determine the biodegradability of some widely used microbicides, these products were dissolved in a mineral solution and synthetic sewage and their breakdown was followed under aerobic and anaerobic conditions. The results indicated that most biocides will not raise pollution problems provided they are discharged into domestic sewage and subsequently treated by aerobic water purification systems. However, all but a few of the biocides tested failed to be degraded in tests simulating conditions prevailing in oligotrophic aquatic systems or in anaerobic ecosystems.
industry and even the home, antimicrobial agents are commonly used to combat micro-organisms. Eventually these chemicals end up in the waste waters. Consequently, the question arises as to how much these compounds are degraded in the aquatic environments. The biodegradability of organic compounds in general and of microbicides in particular can be determined by a variety of tests (Verstraete & Voets, 1972; Pauli & Franke, 1972). However, biodegradation tests simulate only partially the environments in which the breakdown of biocides occurs. To minimize this drawback, the following working hypotheses were proposed. (1) The concentration of the biocide in the waste waters and in the ecosystems which receive and/or treat these waste waters is generally less than l/lOth of the concentration used during disinfection practices. Consequently, the microbicides have been examined for biodegradation at a concentration of l/lOth of their application range. (2) The possibilities for breakdown under aerobic and anaerobic conditions must be considered. (3) It is important t o determine whether breakdown results from metabolic or from co-metabolic microbial activities. (4) To detect any possible toxic metabolites the effluent should be examined by means of a microbiological test. On the basis of these working hypotheses, each biocide was dissolved in a mineral solution and in synthetic sewage. The breakdown of each biocide was subsequently followed under aerobic and anaerobic conditions and monitored by chemical and biological assays. I N HOSPITALS,
Materials and Methods Test procedures The two aerobic tests were performed according to the OCDE-methods for the biodegradability testing of detergents (Anon., 1971a). In the minimal test (MM-test), the microbicide is dissolved in a mineral solution with the following composition 1671
68
J. P. VOETS ET AL.
(gll): (a) KHzP04, 8.5; KzHP04, 21.75; NazHP04.2H20, 33.4; NH4C1, 1.7; (b) MgS04.7Hz0, 22.5; (c) CaClz, 27.5; (d) FeC13.6H20, 0.25. To prepare 1 I of the MM-medium, add 1 ml of each of the solutions a-d to 1 1 of distilled water. One litre of this solution was incubated in a 2-0 1 Erlenmeyer flask on a rotary shaker (120 rim). The flask was inoculated with 1.0 ml of soil extract and incubated open to the air. To prepare the soil extract, 10 g of a fertile field soil were suspended in 100 ml tap water and gently mixed. The suspension was filtered through a 595-Whatman paper filter, the filtrate being used as inoculum. Evaporation losses were regularly adjusted with distilled water. To detect losses of volatile substances, a control flask containing the sterile-filtered microbicide solution was also incubated. The concentration of the microbicide was monitored at daily intervals. The activated sludge test was done according to the procedure described for the confirmatory test in the OCDE-method (Anon., 1971~).The synthetic sewage had the following composition (mgil): peptone, 160; meat extract, 110; urea, 30; NaCI, 7; CaC12.2H20,4; MgS04.7H20,2; tap water 1 1. The synthetic sewage passed through the aeration vessel at a rate of 1 l/h. To start up the activated sludge, the apparatus was fed during a 2-week period with synthetic sewage devoid of microbicides. During the actual trials, the concentration of the test chemical was monitored daily in the influent and effluent. Since this test measures the biodegradation of the microbicide in a complex organic medium, it is referred to as Organic Medium-test (OM-test). The media used in the two anaerobic tests were the same. However, in the anaerobic MM-test, a 1 1 Erlenmeyer flask was filled up to the rim with the MM-solution. The flask was subsequently incubated at 22” in a vacuum chamber. In the anaerobic OM-test, 1 1 of the OM-medium was inoculated with 1 ml of soil extract and incubated under vacuum according to the procedure described above. The concentration of dissolved oxygen was measured by means of a WTW-oxygen probe. The amount of suspensed solids was determined according to the Standard Methods for the Examination of Water and Wastewater (Anon., 1971b). To determine the concentration of the individual microbicides, the following chemical methods were used : for the formaldehyde derivative, the phenyl hydrazine method reported by Pauli & Franke (1972); for the phenolic compounds, the Camino antipyrine method (Anon., 1971b); for Tego the titrimetric method described in the manufacturer’s manual ; for the quaternary compound, the colorimetric method with bromophenolblue as described in the manufacturer’s manual; for the isothiazol derivatives, extraction with methylene chloride and measurement of the absorbance at 282 nm according to the manufacturer’s technical bulletin (Rohm and Haas Coy. To quantify chlorhexidine, an agar diffusion test with Bacillus subtilis as test organism was used (ICI Pharmaceutical Division). Microbiological analyses To examine the effluent of the activated sludge system for residual toxicity towards bacteria, a modification of the procedure described by Bringmann (1973) was used. The following series of 250 ml flasks were prepared: flask a : 10 ml B +O ml A + 100 ml X; flask b: 10 ml B+30 ml A+70 ml X; flask c: 10 ml B+60 ml A 4 4 0 ml X; flask d: 1 0 m l B + 9 0 m l A + 1 0 m l X ; flaske: 1 0 m l B + 1 0 0 m l A + 0 m l X . ‘A‘standsfor a saline solution, NaCl 8.5 g/l; ‘B’ for a solution of Brain Heart Infusion
DEGRADABILITY OF MICROBICIDES
R-N
N-R R
= CH,CH,OH
(i)
69
CI
(i)
Fig. 1. Products tested giving respectively their commercial and chemical names: (a)Grotan B (Schiilke und Marr), 1,3,5-trihydroxy ethyl hexahydrotriazine; (b) Preventol 0 extra (Bayer), o-phenyl phenol ; (c) Preventol CMK (Bayer), p-chloro-rn-cresol; ( d )Triclosan (CIBA-Geigy), S-chloro-2-(2,4-dichlorophenoxy)phenol ; (e) Dichlorophene (Preventol GD, Bayer), 5,5'-dichloro 2,2'-dihydroxy diphenyl methane; (f)Tego 51 (Th. GoldSchmidt), N-dodecyl di(amino-ethyl)-glycine; (g) WSCP-Busan 77 (Buckrnan Lab.), poly oxyethylene (dimethyl iminio) ethylene (dimethyliminio) ethylene dichloride; (h) Chlorhexidine (ICI), 1,6-di (4-chlorophenyl diguanido) hexane; (i) RH 886 ( R o b and Haas), 5-chloro-2-methyl-4-isothiazolin-3-one. CaCle and 2-methyl-4-isothiazolin3-one. C a C h
(Difco), 7-4 g/l and 'X' for the tests solution, sterile-filtered. To prepare the inoculum, Bacillus subtilis (laboratory strain) was inoculated on to a Brain Heart Infusion agai slant. After incubation at 28" for 16 h, the cells of the slant were suspended in 100 ml sterile saline solution. Each flask was inoculated with 5-0 ml of the latter suspension. A 10 ml aliquot was withdrawn from the flasks a-e, and stored at 4". Then, the flasks were incubated at 28" for 24 h on a rotary shaker (120 rim). After this incubation period, the optical density of the preserved ( o . D . ~ ) and of the incubated solutions ( O . D . ~ )was measured at 550 nm. The cell growth (o.D.Q-o.D.~)was plotted against the percentile of the test solution (or against the actual concentration of the microbiocidal compound). The sensitivity limit is defined as the amount of chemical which inhibits the bacterial growth by 25%. These values for the different biocides were (mg!l): Grotan, 25; Preventol 0 extra, 20; Preventol CMK, 28; Triclosan, 0.6; Dichlorophene, 2; Tego 51, 8; WSCP-Busan 77, I ; Chlorhexidine, 8; RH-886, 3.
J. P.VOETS ET AL.
70
To ascertain if the degradation observed in the aerobic activated sludge was due t o metabolic processes, a sample of sludge was washed twice in sterile MM-medium. Then a decimal dilution series of the sludge was prepared in sterile MM-medium. Three test tubes containing 5 ml of sterile MM-medium, supplemented with the appropriate biocide, were inoculated with 1.0ml of each dilution. The series was incubated 2 weeks at 22". The most probable number (MPN) of microbicide metabolizing bacteria present in the sludge was determined by checking the presence of the microbicide in the test tubes at the end of the incubation period.
Results and Discussion The amounts of the different microbicides degraded after 3 weeks of adaptation are given in Table 1. The formaldehyde releasing compound turns out to be very degradable under aerobic conditions. Under anaerobic conditions and in the absence of
TABLE 1 Degradability of various microbicides in diferent tests ~
~~~
% Degradation Aerobic Working conc. (mgil)
1' Formaldehyde releasing compounds 1,3,5-trihydroxy ethyl hexahydrotriazine 2" Phenolic compounds o-phenyl phenol p-chloro-m-cresol 53' dichloro 2,2'dihydroxy diphenylmethane
,
A
I
Anaerobic
Degradation of Microbicides under Different Environmental Conditions
J. P. VOETS, P. PIPYN, P. V A N LANCKER AND W. VERSTRAETE laboratory of General and Industrial Microbiology, State University of Gent, Coupure 533, 9000 Gent, Belgium Received 23 June 1975 and accepted 2 September 1975 To determine the biodegradability of some widely used microbicides, these products were dissolved in a mineral solution and synthetic sewage and their breakdown was followed under aerobic and anaerobic conditions. The results indicated that most biocides will not raise pollution problems provided they are discharged into domestic sewage and subsequently treated by aerobic water purification systems. However, all but a few of the biocides tested failed to be degraded in tests simulating conditions prevailing in oligotrophic aquatic systems or in anaerobic ecosystems.
industry and even the home, antimicrobial agents are commonly used to combat micro-organisms. Eventually these chemicals end up in the waste waters. Consequently, the question arises as to how much these compounds are degraded in the aquatic environments. The biodegradability of organic compounds in general and of microbicides in particular can be determined by a variety of tests (Verstraete & Voets, 1972; Pauli & Franke, 1972). However, biodegradation tests simulate only partially the environments in which the breakdown of biocides occurs. To minimize this drawback, the following working hypotheses were proposed. (1) The concentration of the biocide in the waste waters and in the ecosystems which receive and/or treat these waste waters is generally less than l/lOth of the concentration used during disinfection practices. Consequently, the microbicides have been examined for biodegradation at a concentration of l/lOth of their application range. (2) The possibilities for breakdown under aerobic and anaerobic conditions must be considered. (3) It is important t o determine whether breakdown results from metabolic or from co-metabolic microbial activities. (4) To detect any possible toxic metabolites the effluent should be examined by means of a microbiological test. On the basis of these working hypotheses, each biocide was dissolved in a mineral solution and in synthetic sewage. The breakdown of each biocide was subsequently followed under aerobic and anaerobic conditions and monitored by chemical and biological assays. I N HOSPITALS,
Materials and Methods Test procedures The two aerobic tests were performed according to the OCDE-methods for the biodegradability testing of detergents (Anon., 1971a). In the minimal test (MM-test), the microbicide is dissolved in a mineral solution with the following composition 1671
68
J. P. VOETS ET AL.
(gll): (a) KHzP04, 8.5; KzHP04, 21.75; NazHP04.2H20, 33.4; NH4C1, 1.7; (b) MgS04.7Hz0, 22.5; (c) CaClz, 27.5; (d) FeC13.6H20, 0.25. To prepare 1 I of the MM-medium, add 1 ml of each of the solutions a-d to 1 1 of distilled water. One litre of this solution was incubated in a 2-0 1 Erlenmeyer flask on a rotary shaker (120 rim). The flask was inoculated with 1.0 ml of soil extract and incubated open to the air. To prepare the soil extract, 10 g of a fertile field soil were suspended in 100 ml tap water and gently mixed. The suspension was filtered through a 595-Whatman paper filter, the filtrate being used as inoculum. Evaporation losses were regularly adjusted with distilled water. To detect losses of volatile substances, a control flask containing the sterile-filtered microbicide solution was also incubated. The concentration of the microbicide was monitored at daily intervals. The activated sludge test was done according to the procedure described for the confirmatory test in the OCDE-method (Anon., 1971~).The synthetic sewage had the following composition (mgil): peptone, 160; meat extract, 110; urea, 30; NaCI, 7; CaC12.2H20,4; MgS04.7H20,2; tap water 1 1. The synthetic sewage passed through the aeration vessel at a rate of 1 l/h. To start up the activated sludge, the apparatus was fed during a 2-week period with synthetic sewage devoid of microbicides. During the actual trials, the concentration of the test chemical was monitored daily in the influent and effluent. Since this test measures the biodegradation of the microbicide in a complex organic medium, it is referred to as Organic Medium-test (OM-test). The media used in the two anaerobic tests were the same. However, in the anaerobic MM-test, a 1 1 Erlenmeyer flask was filled up to the rim with the MM-solution. The flask was subsequently incubated at 22” in a vacuum chamber. In the anaerobic OM-test, 1 1 of the OM-medium was inoculated with 1 ml of soil extract and incubated under vacuum according to the procedure described above. The concentration of dissolved oxygen was measured by means of a WTW-oxygen probe. The amount of suspensed solids was determined according to the Standard Methods for the Examination of Water and Wastewater (Anon., 1971b). To determine the concentration of the individual microbicides, the following chemical methods were used : for the formaldehyde derivative, the phenyl hydrazine method reported by Pauli & Franke (1972); for the phenolic compounds, the Camino antipyrine method (Anon., 1971b); for Tego the titrimetric method described in the manufacturer’s manual ; for the quaternary compound, the colorimetric method with bromophenolblue as described in the manufacturer’s manual; for the isothiazol derivatives, extraction with methylene chloride and measurement of the absorbance at 282 nm according to the manufacturer’s technical bulletin (Rohm and Haas Coy. To quantify chlorhexidine, an agar diffusion test with Bacillus subtilis as test organism was used (ICI Pharmaceutical Division). Microbiological analyses To examine the effluent of the activated sludge system for residual toxicity towards bacteria, a modification of the procedure described by Bringmann (1973) was used. The following series of 250 ml flasks were prepared: flask a : 10 ml B +O ml A + 100 ml X; flask b: 10 ml B+30 ml A+70 ml X; flask c: 10 ml B+60 ml A 4 4 0 ml X; flask d: 1 0 m l B + 9 0 m l A + 1 0 m l X ; flaske: 1 0 m l B + 1 0 0 m l A + 0 m l X . ‘A‘standsfor a saline solution, NaCl 8.5 g/l; ‘B’ for a solution of Brain Heart Infusion
DEGRADABILITY OF MICROBICIDES
R-N
N-R R
= CH,CH,OH
(i)
69
CI
(i)
Fig. 1. Products tested giving respectively their commercial and chemical names: (a)Grotan B (Schiilke und Marr), 1,3,5-trihydroxy ethyl hexahydrotriazine; (b) Preventol 0 extra (Bayer), o-phenyl phenol ; (c) Preventol CMK (Bayer), p-chloro-rn-cresol; ( d )Triclosan (CIBA-Geigy), S-chloro-2-(2,4-dichlorophenoxy)phenol ; (e) Dichlorophene (Preventol GD, Bayer), 5,5'-dichloro 2,2'-dihydroxy diphenyl methane; (f)Tego 51 (Th. GoldSchmidt), N-dodecyl di(amino-ethyl)-glycine; (g) WSCP-Busan 77 (Buckrnan Lab.), poly oxyethylene (dimethyl iminio) ethylene (dimethyliminio) ethylene dichloride; (h) Chlorhexidine (ICI), 1,6-di (4-chlorophenyl diguanido) hexane; (i) RH 886 ( R o b and Haas), 5-chloro-2-methyl-4-isothiazolin-3-one. CaCle and 2-methyl-4-isothiazolin3-one. C a C h
(Difco), 7-4 g/l and 'X' for the tests solution, sterile-filtered. To prepare the inoculum, Bacillus subtilis (laboratory strain) was inoculated on to a Brain Heart Infusion agai slant. After incubation at 28" for 16 h, the cells of the slant were suspended in 100 ml sterile saline solution. Each flask was inoculated with 5-0 ml of the latter suspension. A 10 ml aliquot was withdrawn from the flasks a-e, and stored at 4". Then, the flasks were incubated at 28" for 24 h on a rotary shaker (120 rim). After this incubation period, the optical density of the preserved ( o . D . ~ ) and of the incubated solutions ( O . D . ~ )was measured at 550 nm. The cell growth (o.D.Q-o.D.~)was plotted against the percentile of the test solution (or against the actual concentration of the microbiocidal compound). The sensitivity limit is defined as the amount of chemical which inhibits the bacterial growth by 25%. These values for the different biocides were (mg!l): Grotan, 25; Preventol 0 extra, 20; Preventol CMK, 28; Triclosan, 0.6; Dichlorophene, 2; Tego 51, 8; WSCP-Busan 77, I ; Chlorhexidine, 8; RH-886, 3.
J. P.VOETS ET AL.
70
To ascertain if the degradation observed in the aerobic activated sludge was due t o metabolic processes, a sample of sludge was washed twice in sterile MM-medium. Then a decimal dilution series of the sludge was prepared in sterile MM-medium. Three test tubes containing 5 ml of sterile MM-medium, supplemented with the appropriate biocide, were inoculated with 1.0ml of each dilution. The series was incubated 2 weeks at 22". The most probable number (MPN) of microbicide metabolizing bacteria present in the sludge was determined by checking the presence of the microbicide in the test tubes at the end of the incubation period.
Results and Discussion The amounts of the different microbicides degraded after 3 weeks of adaptation are given in Table 1. The formaldehyde releasing compound turns out to be very degradable under aerobic conditions. Under anaerobic conditions and in the absence of
TABLE 1 Degradability of various microbicides in diferent tests ~
~~~
% Degradation Aerobic Working conc. (mgil)
1' Formaldehyde releasing compounds 1,3,5-trihydroxy ethyl hexahydrotriazine 2" Phenolic compounds o-phenyl phenol p-chloro-m-cresol 53' dichloro 2,2'dihydroxy diphenylmethane
,
A
I
Anaerobic
