- Email: [email protected]
Determination of biological degradability of organic substances
14ater Re~eurch Voi. 10. pp 23i to 235 Pergamon Press 19~6. Printed in Great Britain
DETERMINATION OF BIOLOGICAL DEGRADABILITY OF ORGANIC SUBSTANCES P. P11 IER
Department of Water Technology and Environmental Engineering, Prague Institute of Chemical Technology, Prague 6, C.S.S.R.
(Received 29 July 1975) Abstract--The author carried out experiments on the degree and rate of biological degradation of 123 organic compounds with respect to the decrease of organic substance in terms of COD. The organic substances were a sole source of carbon for the microbes of the inoculum, adapted activated sludge being the inoculum. The rate of degradation was. expressed in terms of mg of COD removed by a g of the initial dry matter of inoculum h- t. The degradabilities of 94 aromatic, 15 hydroaromatic, and 14 aliphatic compounds are tabulated.
INTRODUCTION
Biological degradability and toxicity of organic substances are two basic criteria determining their behaviour in natural environment and during the biological treatment of waste waters. From these two points of view organic substances can be divided into the following four groups: I. biologically stances, 2. biologically 3. biologically stances, 4. biologically stances.
degradable
and
non-toxic sub-
degradable and toxic substances, non-degradable and non-toxic subnon-degradable
and toxic sub-
The tests and methods for the determination of biological degradability have been discussed by many authors (Fischer, 1973; Ludzack and Ettinger, 1963; Pitter, 1971). It is usually impossible to find out by chemical or physico-chemical analytical methods if the substance tested has been fully mineralized. Absolute evaluation of degradability according to BOD is difficult since the ratio between oxidation and the synthesis of new biomass differs in some organic substances. Sometimes the oxidation or synthesis of biomass can be inhibited by the tested substance. Besides that also nitrification can have a negative effect. The evaluation of degradability in dependence on the decrease of organic carbon from the biological medium gives the best image of the total amount of removable organic substrate. The degree and rate of degradation can be reliably quantitatively expressed and compared. The degradability of various phenols with respect to their oxidability by potassium permanganate was studied by Meissner as early as 1958. The determination of COD decrease in the evaluation of degradability was used first by Konecky (1963), and later by Pitter (1968) and Janicke (1968). On the basis of experience obtained the Department of Water Technology and Environmental Engineering has developed a standard test for the comparison of biological degradability of organic substances.
Compounds of the first group are non-objectionable. Substances of the second group are, after sufficient dilution, disposed of by natural artificial biological processes. Transfer of compounds of the third, and especially of the fourth, group in the environment should be limited or rendered entirely impossible. It is possible to distinguish about four degrees of degradation (Anon, 1967): primary, partial, acceptable, and total degradation. Partial destruction of molecules of surface-active substances, leading to the loss of surface activity and, consequently, of frothiness as welt, can be considered to be acceptable decomposition. However, the achievement of only partial, although acceptable, biological degradation of organic substances is undesirable. The environment is continually loaded with the remaining fragments Specimen of a standard test for determinin# biological of the original molecules, their long term effects being degradability usually unknown. The test was performed in a batch system. The An easy and complete degradation of organic sub- tested substance is dissolved in a beaker in a biologistances, which come to the environment from in- cal medium in a concentration corresponding to dustry and households, to CO2, H20 phosphates and 200mg 1- I COD. The tested substance is a sole ammoniacal nitrogen, is one of the main requirements source of organic carbon for the microbes of the infor the maintenance of the biological equilibrium in oculum. To the biological medium such amount of nature. thickened adapted activated sludge is added to make 231
1:2
P PIrTER
dr~ matter of the inocuium l(~)mg i . The beaker ~s placed in a dark room ~ith a roughl 3 constant temperature of 20 _ 3 C on an electromagnetic stirrer. The initial value of COD or organic carbon of the liquid phase are determined. Samples. filtered or centrifuged before analysis, are taken at suitable inter~als. The decrease of the tested substance in the liquid phase is evaluated by determining COD or organic carbon. The rest, Its are compared with those of a blank test and standard compound decomposition. With the degree of degradation also the average specific rate of degradation is determined, expressed in terms of mg COD (or organic carbon) removed by a g a m m e of dry matter of the activated sludge per hour. Biological medium. In cq. 800 ml of distilled water solutions of calcium chloride t27"5 g CaCI2 in I 1. distilled waterl, magnesium sulphate (22.5 g MgSO.~: 7 H20 in I 1. distilled water), and ferric chloride (0"25 g FeCI3; 6 H20 in 11. distilled waterl are added in t-ml portions each. Then 5 ml ammonium sulphate solution [10g (NH~):SO.~ in 11. distilled water-], 20ml of a phosphate buffer of pH 7.2 (8.5 g KH,,PO,~, 21.8g KaHPO., and 44.7g Na:HPOa: 12 H : O in 1 1. distilled water), and 100ml of tap water for securing the content of trace elements, are added. The solution thus prepared is made up to 1000ml with distilled water. lnoe~dum. The adaptation of the activated sludge is made in the following way. Activated sludge taken from a sewage plant is cultivated in a 1000ml volu-metric cylinder. The mixture is aerated with pressure air. Every day 200 ml of the mixture is driven off so that the sludge age is 5 days. After driving off the 200ml of the mixture aeration is interrupted, and after sedimentation ca. 6 0 0 m l of the liquid phase is driven off. The residue (200 ml of the thickened activated sludge) is diluted with tap water to the volume of ca. 800 ml and 600 mg 1-' of starch or glucose, 600 mg 1 - ' of peptone, 25 ml of a phosphate buffer pH 7.2, and the solution of the tested compound are added. Then the mixture in the cylinder is made up to 1000ml with tap water and aerated for 23 h (the recirculation ratio is 0-25). After this period the procedure is repeated. The concentration of the tested substance is gradually increased so that after 20 days of adaptation it reaches the equivalent value of 200rag I - ' COD. During this period an occasional biological analysis is made and the change of biocenosis is evaluated. In case the substance is a toxic one, the sludge must be adapted at lower initial concentrations, and the concentration of the compound in the actual experiment on degradability is to be decreased equally. Procedure. To 1000-1500ml of the biological medium such amount of the solution of the substance tested is added that the initial COD is 200mg 1Then such an amount of the adapted activated sludge, washed and thickened by sedimentation, is dosed to the medium that the concentration of the dry matter
is 100mg I ~. Simultaneousl,, a blank test is prepared, or possib[? also a test with a standard compound, to ~eril'3 the activit3 of the inoculum tglucose, phenol etc.). With volatile substances a test without the inoculum is to be carried out as well in order to differentiate the actual biological de~adation and the losses due to mere volatalization. From the beakers 50-80ml of the mixture are taken, and after filtration through a filter paper of medium porosity the initial values of COD or organic carbon of the liquid phase are determined. If the filtrates are turbid the liquid phase must be separated by centrifuging or with membrane ultrafilters with average pore size 0.6-1.0 :zm. The initial levels of the mixture in the beaker is marked either with pencil on the glass or by means of a strip of mm paper stuck to the outer wall of the beaker. Afterwards the beakers are placed in a dark room with a temperature 20 + 3:C on magnetic stirrers. The oxygenation capacity of this device was ca. I l m g of oxygen h - x I - t at 800 rev rainAt conveniently chosen time intervals always ca. 50-80 ml of the sample are taken for analysis, When the time intervals are longer the potential losses due to evaporation should be made up with distilled water prior to each sampling. The time intervals between the individual samplings depend on the rate of degradation. With very readily degradable substances the process is completed as early as in 24 h. Generally it is sufficient to take samples once or twice a day. The values of the blank test are subtracted. The experiment is carried out till there is no decrease of COD. After that time the total percentage of COD removed and the rate of degradation are evaluated. If under the given experimental conditions already in 120 h of incubation over 90°,/0 of the initial COD is removed (the degradation rate being over 15 mg of COD g- t h - t), the substance tested can be considered to be biologically readily decomposable. When even after 20 days of incubation there is no complete biological degradation, the experiments are ~°~ "-''-'~e -'-'-'-"
°" 4
•
•
.\
[ L o
7
g o
ioo 2,9o h Fig. 1. The course of the biological degradation of glycerol (I), diethanolamine (2), o-nitrophenol (3), and 3,5-dinitrosalic?lic acid (4).
233
Biological degadability of organic substances Table I. Biological degradability of aliphatic compounds Percent removed (based upon COD)
Compound Ammonium oxalate n-butanol See. butanol Tert. butanol 1,4-Butanediol Diethylene glycol Diethanolamine Ethylene diamine Ethylene glycol Glycerol Glucose n-Propanol lso-Propanol Triethylene giycol
92.5 98'8 98"5 98"5 98.7 95.0 97.0 97.5 96'8 98.7 98"5 98'8 99.0 97.7
repeated at lower initial concentration of the substance tested. With very low initial values of COD or organic carbon the degradation is to be studied using a suitable structural--analytical methods. The course of the biological degradation of glycerol, diethanolamine, o-nitrophenol, and 3,5-dinitrosalicylic acid under described experimental conditions is summarized in Fig. 1. An example of evaluation. Compound; p-nitrophenol. Procedure; a batch test in an open system. The compound was the sole source of organic carbon for the microbes of the inoculum. Degradation was studied by determining COD (organic carbon). The pH of the biological medium was 7.2. Initial concentration; 125 = 200rag 1-t COD. Inoculum; Activated sludge, adapted for 20 days, fed by glucose and peptone with simultaneous addition of the substance tested in the concentration up to 125 mg -~. The sludge age was 5 days. Concentration of the inoculum; 100mg 1-~. Time of the experiment; 120h. Average temperature during the experiment; 20°C. Degree of degradation; 95~ with respect to COD. Average rate of the degradation; 15-8 mg of COD gh- 1. Evaluation; p-Nitrophenol belongs to substances
Rate of biodegradation (mg COD g- t h- i) 9'3 84-0 55~3 304) 40.0 13.7 19.5 9.8 41.7 85"0 180'0 71.0 52.0 27.5
which are biologically readily degradable. During adaptation of the inoculum no change in the biocenosis of the activated sludge was observed. RESULTS AND DISCUSSION
The results of experiments with biological degradability of aliphatic, hydroaromatic, and aromatic compounds are summarized in Tables 1-3, respectively. To the compounds which are biologically hard to decompose belong: pyrogallol, metol, 3,4-dimetylaniline, N-phenylanthranilic acid, 2-chloro-4-nitrophenol, 2,4,6-trinitrophenol, 3,5-dinitrobenzoic acid, 3,5-dinitrosalicylic acid, nitroanilines, o-phenylendiamine, m-phenylendiamine, aminophenolsulphonic acid, 1-naphthylamine, 1-naphthylamine-6-sulphonic acid, m-benz, nedisulphonic acid, 2,5-dinitrophenol, 2,6-dinitrophenol, 1,3-dinitrobenzene, and 1,4-dinitrobenzene. With these compounds only a low or zero removal of COD was achieved. The discussion of the relations between structure and biolobical degradability of the organic substances studied is contained in the author's earlier papers (Pitter, 1974; 1975). It gives examples of explanation
Table 2. Biological degradability of cycloaliphatic compounds Compound Borneol Caprolactam Cyclohexanol Cyclopentanol Cyclohexanone Cyclopcntanone Cyclohexanolone 1,2-Cyclohexanediol Dimethylcyclohexanol 4-Methylcyclohexanol 4- Methylcyclohexanone Menthol Tetrahydrofurfuryl alcohol Tetrahydrophthalimide Tetrahydrophthalic acid
Percent removed (based upon COD)
Rate of biodegradation (rag COD g-t h-t)
90-3 94.3 96"0 97.0 96-0 95-4 92-4 95.0 92"3 94-0 96.7 95.1 96-I 0 0
8'9 16-0 28"0 55"0 30-0 57.0 51.5 66.0 21'6 40-0 62.5 17.7 40.0 ---
P, PITTER
Table 3. Biological de~adability of aromatic compounds Compound Aniline Aminophenolsulphonic acid Acetanilide p-Aminoacetanilide o-Aminotoluene m-Aminotoluene p-Aminotoluene o-Aminobenzoic acid m-Aminobenzoic acid p-Aminobenzoic acid o-Aminophenol m-Aminophenol p-Aminophenol Benzenesulphonic acid m-Benzenedisulphonic acid Benzaldehyde Benzoic acid o-Cresol m-Cresol p-Cresol D-Chloramphenicol o-Chlorophenol p-Chlorophenol o-Chloroaniline m-Chloroaniline p-Chloroaniline 2-Chloro-4-nit rophenol 2,4-Dichtorophenol 1,3-Dinitrobenzene 1,4-Dinitrobenzene 2,3-Dimethylphenol 2,4-Dimethylphenol .~ 3,4-Dimethylphenol 3,5-Dimethylphenol 2,5-Dimethylphenol 2,6-Dimethylphenol 3,4-Dimethylaniline 2,3-Dimethylaniline 2,5-Dimethylanitine 2,4-Diaminophenol 2,5-Dinitrophenol 2,6-Dinitrophenol 2,4-Dinitrophenol 3,5-Dinitrobenzoic acid 3,5-Dinitrosalicylic acid Furfuryl alcohol Furfurylaldehyde Gallic acid Gentisic acid p-Hydroxybenzoic acid Hydroquinone lsophthalic acid Metol Naphtoic acid
l-Naphthol 1-Naphthylamine l-Naphthalenesulfonic acid
l-Naphthol-2-sulphonicacid l-Naphthylamine-6-sulphonic acid 2-Naphthol p-Nitroacetophenone Nitrobenzene o-Nitrophenol m-Nitrophenol p-Nitrophenol o-Nitroluene m-Nitrotoluene p-Nitrotoluene
Percent remo;ed lbased upon CODI
Rate of biodegadation Imp,. COD _-"-t h -~}
94.5 64.6 94.5 93.0 97-7 97.7 97.7 97.5 97.5 96.2 95.0 90-5 87"0 98-5 63.5 99.0 99.0 95.0 95.5 96.0 86.2 95.6 96-0 98-0 97.2 96.5 71.5 98.0 0 0 95.5 94.5 97.5 89.3 94.5 94-3 76.0 96.5 96.5 83.0
19.0 7.1 14.7 11.3 15.I 30-0 20.0 27-1 7.0 12.5 21.l 10-6 16.7 10.6 3.4 119.0 88.5 54-0 55.0 55-0 3.3 25-0 11.0 16.7 62 5.7 5-3 10.5 35-0 28-2 13.4 11.i 10.6 9.0 30.0 12.7 3.6 12.0 see note 1 see note 1
85.0 50.0 0 97.3 96.3 90.5 97.6 98.7 90'0 95-0 594 90.2 92.1 0 90.5 9 l'0 0 89.0 98.8 98.0 97.0 95.0 95-0 98.0 98.5 98'0
6.0 41.0 37"0 20.0 80-0 100.0 54.2 76.0 0'8 15.5 38.4 0 18.0 18.0 0 39.2 52 14-0 14.0 17.5 17.5 32-5 21-0 32.5
Biological degradability of organic substances
235
Table 3 (cont.) Compound
Percent removed (based upon COD)
o-Nitrobenzaldehyde m-Nitrobenzaldehyde p,Nitrobenzaldehyde o-Nitrobenzoic acid m-Nitrobenzoic acid p-Nitrobenzoic acid o-Nitroaniline (see note 2) m-Nitroaniline (see note 2) p-Nitroaniline (see note 2) Phthalimide Phthalic acid Phenol Phlorogiucinol N-Phenylanthranilic acid o-Phenylendiamine (see note 3) m-Phenylendiamine (see note 3) p-Phenylendiamine (see note 3) Pyrocatechol Pyrogallol Resorcinol Salicylic acid Sulphosalicylic acid Sulphanilic acid Thymol p-Toluenesulphonic acid 2,4,6-Trinitrophenol
97.0 94.0 97.0 93.4 93-4 92-0 0 0 0 96.2 96"8 98.5 92"5 28"0 33'0 60"0 80"0 96"0 40.0 90'0 98'8 98"5 95.0 94'6 98.7 0
Rate of biodem'adation (mg COD g-~ h-t) 13-8 10-0 13-8 20-0 7-0 19-7 ---20-8 78-4 80-0 22"1 ----55-5 -57-5 95"0 11"3 4.0 15-6 8-4 --
Note 1. 2,5 and 2,6-dinitrophenol were at higher concentrations not degraded. 2.6-Dinitrophenol was at lower concentrations decomposed with long adapted activated sludge (40 days). 2,5-Dinitrophenol was biochemically stable. Note 2. The degradation of nitroanilines was determined photometrically in the concentration range from 25 to 30 trig l - t . Note 3. The degradation of phenylenediamines was determined photometrically in the concentration range from 25 to 30 mg l- t. p-Phenylenediamine was comparatively well degradable. from the point of view of classical electronic theory of mechanisms of organic reactions and from the point of view of findings of quantum chemistry. On the basis of the knowledge obtained the factors affecting biological degradability can be divided into three groups: I. physico-chemical factors (temperature, solubility, degree of dispersion of the compound in the medium, pH, dissolved oxygen), 2. biological factors (history of the microbial culture, its age, manner and time of its adaptation, toxicity of the compound, effect of other substrates) and 3. chemical factors (size of molecule, length of chain, kind, number and position of substituents in the molecule, stereochemistry).
REFERENCES
Anon (1967) Required characteristics and measurement of biodegradability. J. War. Pollut. Control Fed. 39, 1232-1235. Fischer W. K. (1973) Priifung der biologischen Abbaubarkeit yon synthetischen Verbindungen. z. B. Detergentien. Vom Wass. 40, 305-333.
Janicke W. (1968) Indirekte Ermittlung des biologischen Abbaugrades synthetischer organischer Verbindungen dutch Bestimmung der Oxydierbarkeiten. Gesundheitsing 89, 309-314. Konecky M. S., Kelly R. J., Symons J. M., McCarty P. L. (1963) The determination of the biodegradability of detergents. ESSO Research Biodegradation Test. 36th Ann. Meeting of War. PoUut. Control Fed. Seattle, Washington. Ludzack F. J. and Ettinger M. B. (1963) Estimating biodegradability and treatability of organic water pollutants. Biotechnol. Bioengng 5, 309-329. Meissner B. 0958) Zum biologischen Abbau organischer Substanzen. Wasserwirtschaft u. Wasserrechnik 8, 483-489. Pitter P. {1968) Surface active agents in waste waters. XII. Evaluation of surfactant biodegradability by the COD technique and ultraviolet spectra. Sb. vys. ~k. chera.-technol. c Pra=e F 14, 7-17. Pitter P. (1971) Determination of the biological degradability of organic substances. Chem. listy 65, 897-921 (In Czech). Pitter P. (1974) Relation between structure and biological degradability of organic compounds. Sb. vys. ~k. chem.technol, v Praze F 19, 43--106 {In Czech). Pitter P. 0976) Relations between the structure of organic compounds and their biological degradability. Cheraick~ prdtmysl 25. No. I. (In Czech). Swisher R. D. (1970) Surfactant biodegradation. Dekker, New York.
DETERMINATION OF BIOLOGICAL DEGRADABILITY OF ORGANIC SUBSTANCES P. P11 IER
Department of Water Technology and Environmental Engineering, Prague Institute of Chemical Technology, Prague 6, C.S.S.R.
(Received 29 July 1975) Abstract--The author carried out experiments on the degree and rate of biological degradation of 123 organic compounds with respect to the decrease of organic substance in terms of COD. The organic substances were a sole source of carbon for the microbes of the inoculum, adapted activated sludge being the inoculum. The rate of degradation was. expressed in terms of mg of COD removed by a g of the initial dry matter of inoculum h- t. The degradabilities of 94 aromatic, 15 hydroaromatic, and 14 aliphatic compounds are tabulated.
INTRODUCTION
Biological degradability and toxicity of organic substances are two basic criteria determining their behaviour in natural environment and during the biological treatment of waste waters. From these two points of view organic substances can be divided into the following four groups: I. biologically stances, 2. biologically 3. biologically stances, 4. biologically stances.
degradable
and
non-toxic sub-
degradable and toxic substances, non-degradable and non-toxic subnon-degradable
and toxic sub-
The tests and methods for the determination of biological degradability have been discussed by many authors (Fischer, 1973; Ludzack and Ettinger, 1963; Pitter, 1971). It is usually impossible to find out by chemical or physico-chemical analytical methods if the substance tested has been fully mineralized. Absolute evaluation of degradability according to BOD is difficult since the ratio between oxidation and the synthesis of new biomass differs in some organic substances. Sometimes the oxidation or synthesis of biomass can be inhibited by the tested substance. Besides that also nitrification can have a negative effect. The evaluation of degradability in dependence on the decrease of organic carbon from the biological medium gives the best image of the total amount of removable organic substrate. The degree and rate of degradation can be reliably quantitatively expressed and compared. The degradability of various phenols with respect to their oxidability by potassium permanganate was studied by Meissner as early as 1958. The determination of COD decrease in the evaluation of degradability was used first by Konecky (1963), and later by Pitter (1968) and Janicke (1968). On the basis of experience obtained the Department of Water Technology and Environmental Engineering has developed a standard test for the comparison of biological degradability of organic substances.
Compounds of the first group are non-objectionable. Substances of the second group are, after sufficient dilution, disposed of by natural artificial biological processes. Transfer of compounds of the third, and especially of the fourth, group in the environment should be limited or rendered entirely impossible. It is possible to distinguish about four degrees of degradation (Anon, 1967): primary, partial, acceptable, and total degradation. Partial destruction of molecules of surface-active substances, leading to the loss of surface activity and, consequently, of frothiness as welt, can be considered to be acceptable decomposition. However, the achievement of only partial, although acceptable, biological degradation of organic substances is undesirable. The environment is continually loaded with the remaining fragments Specimen of a standard test for determinin# biological of the original molecules, their long term effects being degradability usually unknown. The test was performed in a batch system. The An easy and complete degradation of organic sub- tested substance is dissolved in a beaker in a biologistances, which come to the environment from in- cal medium in a concentration corresponding to dustry and households, to CO2, H20 phosphates and 200mg 1- I COD. The tested substance is a sole ammoniacal nitrogen, is one of the main requirements source of organic carbon for the microbes of the infor the maintenance of the biological equilibrium in oculum. To the biological medium such amount of nature. thickened adapted activated sludge is added to make 231
1:2
P PIrTER
dr~ matter of the inocuium l(~)mg i . The beaker ~s placed in a dark room ~ith a roughl 3 constant temperature of 20 _ 3 C on an electromagnetic stirrer. The initial value of COD or organic carbon of the liquid phase are determined. Samples. filtered or centrifuged before analysis, are taken at suitable inter~als. The decrease of the tested substance in the liquid phase is evaluated by determining COD or organic carbon. The rest, Its are compared with those of a blank test and standard compound decomposition. With the degree of degradation also the average specific rate of degradation is determined, expressed in terms of mg COD (or organic carbon) removed by a g a m m e of dry matter of the activated sludge per hour. Biological medium. In cq. 800 ml of distilled water solutions of calcium chloride t27"5 g CaCI2 in I 1. distilled waterl, magnesium sulphate (22.5 g MgSO.~: 7 H20 in I 1. distilled water), and ferric chloride (0"25 g FeCI3; 6 H20 in 11. distilled waterl are added in t-ml portions each. Then 5 ml ammonium sulphate solution [10g (NH~):SO.~ in 11. distilled water-], 20ml of a phosphate buffer of pH 7.2 (8.5 g KH,,PO,~, 21.8g KaHPO., and 44.7g Na:HPOa: 12 H : O in 1 1. distilled water), and 100ml of tap water for securing the content of trace elements, are added. The solution thus prepared is made up to 1000ml with distilled water. lnoe~dum. The adaptation of the activated sludge is made in the following way. Activated sludge taken from a sewage plant is cultivated in a 1000ml volu-metric cylinder. The mixture is aerated with pressure air. Every day 200 ml of the mixture is driven off so that the sludge age is 5 days. After driving off the 200ml of the mixture aeration is interrupted, and after sedimentation ca. 6 0 0 m l of the liquid phase is driven off. The residue (200 ml of the thickened activated sludge) is diluted with tap water to the volume of ca. 800 ml and 600 mg 1-' of starch or glucose, 600 mg 1 - ' of peptone, 25 ml of a phosphate buffer pH 7.2, and the solution of the tested compound are added. Then the mixture in the cylinder is made up to 1000ml with tap water and aerated for 23 h (the recirculation ratio is 0-25). After this period the procedure is repeated. The concentration of the tested substance is gradually increased so that after 20 days of adaptation it reaches the equivalent value of 200rag I - ' COD. During this period an occasional biological analysis is made and the change of biocenosis is evaluated. In case the substance is a toxic one, the sludge must be adapted at lower initial concentrations, and the concentration of the compound in the actual experiment on degradability is to be decreased equally. Procedure. To 1000-1500ml of the biological medium such amount of the solution of the substance tested is added that the initial COD is 200mg 1Then such an amount of the adapted activated sludge, washed and thickened by sedimentation, is dosed to the medium that the concentration of the dry matter
is 100mg I ~. Simultaneousl,, a blank test is prepared, or possib[? also a test with a standard compound, to ~eril'3 the activit3 of the inoculum tglucose, phenol etc.). With volatile substances a test without the inoculum is to be carried out as well in order to differentiate the actual biological de~adation and the losses due to mere volatalization. From the beakers 50-80ml of the mixture are taken, and after filtration through a filter paper of medium porosity the initial values of COD or organic carbon of the liquid phase are determined. If the filtrates are turbid the liquid phase must be separated by centrifuging or with membrane ultrafilters with average pore size 0.6-1.0 :zm. The initial levels of the mixture in the beaker is marked either with pencil on the glass or by means of a strip of mm paper stuck to the outer wall of the beaker. Afterwards the beakers are placed in a dark room with a temperature 20 + 3:C on magnetic stirrers. The oxygenation capacity of this device was ca. I l m g of oxygen h - x I - t at 800 rev rainAt conveniently chosen time intervals always ca. 50-80 ml of the sample are taken for analysis, When the time intervals are longer the potential losses due to evaporation should be made up with distilled water prior to each sampling. The time intervals between the individual samplings depend on the rate of degradation. With very readily degradable substances the process is completed as early as in 24 h. Generally it is sufficient to take samples once or twice a day. The values of the blank test are subtracted. The experiment is carried out till there is no decrease of COD. After that time the total percentage of COD removed and the rate of degradation are evaluated. If under the given experimental conditions already in 120 h of incubation over 90°,/0 of the initial COD is removed (the degradation rate being over 15 mg of COD g- t h - t), the substance tested can be considered to be biologically readily decomposable. When even after 20 days of incubation there is no complete biological degradation, the experiments are ~°~ "-''-'~e -'-'-'-"
°" 4
•
•
.\
[ L o
7
g o
ioo 2,9o h Fig. 1. The course of the biological degradation of glycerol (I), diethanolamine (2), o-nitrophenol (3), and 3,5-dinitrosalic?lic acid (4).
233
Biological degadability of organic substances Table I. Biological degradability of aliphatic compounds Percent removed (based upon COD)
Compound Ammonium oxalate n-butanol See. butanol Tert. butanol 1,4-Butanediol Diethylene glycol Diethanolamine Ethylene diamine Ethylene glycol Glycerol Glucose n-Propanol lso-Propanol Triethylene giycol
92.5 98'8 98"5 98"5 98.7 95.0 97.0 97.5 96'8 98.7 98"5 98'8 99.0 97.7
repeated at lower initial concentration of the substance tested. With very low initial values of COD or organic carbon the degradation is to be studied using a suitable structural--analytical methods. The course of the biological degradation of glycerol, diethanolamine, o-nitrophenol, and 3,5-dinitrosalicylic acid under described experimental conditions is summarized in Fig. 1. An example of evaluation. Compound; p-nitrophenol. Procedure; a batch test in an open system. The compound was the sole source of organic carbon for the microbes of the inoculum. Degradation was studied by determining COD (organic carbon). The pH of the biological medium was 7.2. Initial concentration; 125 = 200rag 1-t COD. Inoculum; Activated sludge, adapted for 20 days, fed by glucose and peptone with simultaneous addition of the substance tested in the concentration up to 125 mg -~. The sludge age was 5 days. Concentration of the inoculum; 100mg 1-~. Time of the experiment; 120h. Average temperature during the experiment; 20°C. Degree of degradation; 95~ with respect to COD. Average rate of the degradation; 15-8 mg of COD gh- 1. Evaluation; p-Nitrophenol belongs to substances
Rate of biodegradation (mg COD g- t h- i) 9'3 84-0 55~3 304) 40.0 13.7 19.5 9.8 41.7 85"0 180'0 71.0 52.0 27.5
which are biologically readily degradable. During adaptation of the inoculum no change in the biocenosis of the activated sludge was observed. RESULTS AND DISCUSSION
The results of experiments with biological degradability of aliphatic, hydroaromatic, and aromatic compounds are summarized in Tables 1-3, respectively. To the compounds which are biologically hard to decompose belong: pyrogallol, metol, 3,4-dimetylaniline, N-phenylanthranilic acid, 2-chloro-4-nitrophenol, 2,4,6-trinitrophenol, 3,5-dinitrobenzoic acid, 3,5-dinitrosalicylic acid, nitroanilines, o-phenylendiamine, m-phenylendiamine, aminophenolsulphonic acid, 1-naphthylamine, 1-naphthylamine-6-sulphonic acid, m-benz, nedisulphonic acid, 2,5-dinitrophenol, 2,6-dinitrophenol, 1,3-dinitrobenzene, and 1,4-dinitrobenzene. With these compounds only a low or zero removal of COD was achieved. The discussion of the relations between structure and biolobical degradability of the organic substances studied is contained in the author's earlier papers (Pitter, 1974; 1975). It gives examples of explanation
Table 2. Biological degradability of cycloaliphatic compounds Compound Borneol Caprolactam Cyclohexanol Cyclopentanol Cyclohexanone Cyclopcntanone Cyclohexanolone 1,2-Cyclohexanediol Dimethylcyclohexanol 4-Methylcyclohexanol 4- Methylcyclohexanone Menthol Tetrahydrofurfuryl alcohol Tetrahydrophthalimide Tetrahydrophthalic acid
Percent removed (based upon COD)
Rate of biodegradation (rag COD g-t h-t)
90-3 94.3 96"0 97.0 96-0 95-4 92-4 95.0 92"3 94-0 96.7 95.1 96-I 0 0
8'9 16-0 28"0 55"0 30-0 57.0 51.5 66.0 21'6 40-0 62.5 17.7 40.0 ---
P, PITTER
Table 3. Biological de~adability of aromatic compounds Compound Aniline Aminophenolsulphonic acid Acetanilide p-Aminoacetanilide o-Aminotoluene m-Aminotoluene p-Aminotoluene o-Aminobenzoic acid m-Aminobenzoic acid p-Aminobenzoic acid o-Aminophenol m-Aminophenol p-Aminophenol Benzenesulphonic acid m-Benzenedisulphonic acid Benzaldehyde Benzoic acid o-Cresol m-Cresol p-Cresol D-Chloramphenicol o-Chlorophenol p-Chlorophenol o-Chloroaniline m-Chloroaniline p-Chloroaniline 2-Chloro-4-nit rophenol 2,4-Dichtorophenol 1,3-Dinitrobenzene 1,4-Dinitrobenzene 2,3-Dimethylphenol 2,4-Dimethylphenol .~ 3,4-Dimethylphenol 3,5-Dimethylphenol 2,5-Dimethylphenol 2,6-Dimethylphenol 3,4-Dimethylaniline 2,3-Dimethylaniline 2,5-Dimethylanitine 2,4-Diaminophenol 2,5-Dinitrophenol 2,6-Dinitrophenol 2,4-Dinitrophenol 3,5-Dinitrobenzoic acid 3,5-Dinitrosalicylic acid Furfuryl alcohol Furfurylaldehyde Gallic acid Gentisic acid p-Hydroxybenzoic acid Hydroquinone lsophthalic acid Metol Naphtoic acid
l-Naphthol 1-Naphthylamine l-Naphthalenesulfonic acid
l-Naphthol-2-sulphonicacid l-Naphthylamine-6-sulphonic acid 2-Naphthol p-Nitroacetophenone Nitrobenzene o-Nitrophenol m-Nitrophenol p-Nitrophenol o-Nitroluene m-Nitrotoluene p-Nitrotoluene
Percent remo;ed lbased upon CODI
Rate of biodegadation Imp,. COD _-"-t h -~}
94.5 64.6 94.5 93.0 97-7 97.7 97.7 97.5 97.5 96.2 95.0 90-5 87"0 98-5 63.5 99.0 99.0 95.0 95.5 96.0 86.2 95.6 96-0 98-0 97.2 96.5 71.5 98.0 0 0 95.5 94.5 97.5 89.3 94.5 94-3 76.0 96.5 96.5 83.0
19.0 7.1 14.7 11.3 15.I 30-0 20.0 27-1 7.0 12.5 21.l 10-6 16.7 10.6 3.4 119.0 88.5 54-0 55.0 55-0 3.3 25-0 11.0 16.7 62 5.7 5-3 10.5 35-0 28-2 13.4 11.i 10.6 9.0 30.0 12.7 3.6 12.0 see note 1 see note 1
85.0 50.0 0 97.3 96.3 90.5 97.6 98.7 90'0 95-0 594 90.2 92.1 0 90.5 9 l'0 0 89.0 98.8 98.0 97.0 95.0 95-0 98.0 98.5 98'0
6.0 41.0 37"0 20.0 80-0 100.0 54.2 76.0 0'8 15.5 38.4 0 18.0 18.0 0 39.2 52 14-0 14.0 17.5 17.5 32-5 21-0 32.5
Biological degradability of organic substances
235
Table 3 (cont.) Compound
Percent removed (based upon COD)
o-Nitrobenzaldehyde m-Nitrobenzaldehyde p,Nitrobenzaldehyde o-Nitrobenzoic acid m-Nitrobenzoic acid p-Nitrobenzoic acid o-Nitroaniline (see note 2) m-Nitroaniline (see note 2) p-Nitroaniline (see note 2) Phthalimide Phthalic acid Phenol Phlorogiucinol N-Phenylanthranilic acid o-Phenylendiamine (see note 3) m-Phenylendiamine (see note 3) p-Phenylendiamine (see note 3) Pyrocatechol Pyrogallol Resorcinol Salicylic acid Sulphosalicylic acid Sulphanilic acid Thymol p-Toluenesulphonic acid 2,4,6-Trinitrophenol
97.0 94.0 97.0 93.4 93-4 92-0 0 0 0 96.2 96"8 98.5 92"5 28"0 33'0 60"0 80"0 96"0 40.0 90'0 98'8 98"5 95.0 94'6 98.7 0
Rate of biodem'adation (mg COD g-~ h-t) 13-8 10-0 13-8 20-0 7-0 19-7 ---20-8 78-4 80-0 22"1 ----55-5 -57-5 95"0 11"3 4.0 15-6 8-4 --
Note 1. 2,5 and 2,6-dinitrophenol were at higher concentrations not degraded. 2.6-Dinitrophenol was at lower concentrations decomposed with long adapted activated sludge (40 days). 2,5-Dinitrophenol was biochemically stable. Note 2. The degradation of nitroanilines was determined photometrically in the concentration range from 25 to 30 trig l - t . Note 3. The degradation of phenylenediamines was determined photometrically in the concentration range from 25 to 30 mg l- t. p-Phenylenediamine was comparatively well degradable. from the point of view of classical electronic theory of mechanisms of organic reactions and from the point of view of findings of quantum chemistry. On the basis of the knowledge obtained the factors affecting biological degradability can be divided into three groups: I. physico-chemical factors (temperature, solubility, degree of dispersion of the compound in the medium, pH, dissolved oxygen), 2. biological factors (history of the microbial culture, its age, manner and time of its adaptation, toxicity of the compound, effect of other substrates) and 3. chemical factors (size of molecule, length of chain, kind, number and position of substituents in the molecule, stereochemistry).
REFERENCES
Anon (1967) Required characteristics and measurement of biodegradability. J. War. Pollut. Control Fed. 39, 1232-1235. Fischer W. K. (1973) Priifung der biologischen Abbaubarkeit yon synthetischen Verbindungen. z. B. Detergentien. Vom Wass. 40, 305-333.
Janicke W. (1968) Indirekte Ermittlung des biologischen Abbaugrades synthetischer organischer Verbindungen dutch Bestimmung der Oxydierbarkeiten. Gesundheitsing 89, 309-314. Konecky M. S., Kelly R. J., Symons J. M., McCarty P. L. (1963) The determination of the biodegradability of detergents. ESSO Research Biodegradation Test. 36th Ann. Meeting of War. PoUut. Control Fed. Seattle, Washington. Ludzack F. J. and Ettinger M. B. (1963) Estimating biodegradability and treatability of organic water pollutants. Biotechnol. Bioengng 5, 309-329. Meissner B. 0958) Zum biologischen Abbau organischer Substanzen. Wasserwirtschaft u. Wasserrechnik 8, 483-489. Pitter P. {1968) Surface active agents in waste waters. XII. Evaluation of surfactant biodegradability by the COD technique and ultraviolet spectra. Sb. vys. ~k. chera.-technol. c Pra=e F 14, 7-17. Pitter P. (1971) Determination of the biological degradability of organic substances. Chem. listy 65, 897-921 (In Czech). Pitter P. (1974) Relation between structure and biological degradability of organic compounds. Sb. vys. ~k. chem.technol, v Praze F 19, 43--106 {In Czech). Pitter P. 0976) Relations between the structure of organic compounds and their biological degradability. Cheraick~ prdtmysl 25. No. I. (In Czech). Swisher R. D. (1970) Surfactant biodegradation. Dekker, New York.