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A critical comparison of respirometric biodegradation tests based on OECD 301 and related test methods
Water Research 37 (2003) 1571–1582
A critical comparison of respirometric biodegradation tests based on OECD 301 and related test methods Peter Reuschenbacha, Udo Paggaa, Uwe Strotmannb,* b
a BASF Aktiengesellschaft, Laboratory of Ecological Studies GV/TC Z570, D-67056 Ludwigshafen, Germany Department of Environmental Technology, Fachhochschule Gelsenkirchen, University of Applied Sciences, Neidenburger Str. 10, D-45877 Gelsenkirchen, Germany
Received 8 January 2002; received in revised form 14 June 2002; accepted 22 October 2002
Abstract Biodegradation studies of organic compounds in the aquatic environment gain important information for the final fate of chemicals in the environment. A decisive role play tests for ready biodegradability (OECD 301) and in this context, the respirometric test (OECD 301F). Two different respirometric systems (Oxitops and Sapromats) were compared and in two of ten cases (diethylene glycol and 2-ethylhexylacrylate) differences were observed indicating that the test systems are not always equivalent. For 2-ethylhexylacrylate and cyclohexanone we could not state differences in the extent of biodegradation with a municipal and industrial inoculum whereas for cyclohexanone the degradation rate was faster with a municipal inoculum. Allylthiourea (ATU) proved to be an effective inhibitor of nitrification processes and did not affect the heterotrophic biodegradation activity. Modelling of biodegradation processes could be successfully performed with a first-order and a modified logistic plot. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Biodegradation; Mathematical modelling; OECD 301 tests; Ready biodegradability; Respirometry
1. Introduction Over past 40 years, the fate of chemical pollutions in the environment has become an important issue. Methods for investigating and monitoring biodegradation processes have been developed [1]. Most efforts have concentrated on the fate of chemicals in the aquatic environment, especially in wastewater treatment processes. Although the elimination of chemicals from the aquatic environment may occur by abiotic processes such as adsorption, hydrolysis and photolysis, the complete conversion of organic chemicals to inorganic products is due to microbial biodegradation processes. During these ultimate biodegradation processes the *Corresponding author. Tel.: +49-209-9596-142; fax: +49209-9596-144. E-mail address: [email protected] (U. Strotmann).
organic matter is converted into CO2, H2O, inorganic salts, microbial biomass and organic metabolites [2]. Nearly 20 years ago, in 1981 the OECD first published its guidelines for testing the biodegradation of chemicals which were updated 1993 [3]. In the mean time, also a number of biodegradation standards of the International Organisation of Standardisation (ISO) [4], which are to some extent similar to the OECD guidelines, have been created. An overview gives ISO 15462 and Pagga [5]. In the OECD guidelines biodegradation tests are divided into three principal categories: tests for ready biodegradability, tests for inherent biodegradability and simulation tests. The most important tests for practical use are the tests for ready biodegradability. These are the most stringent tests, offering only limited opportunities for biodegradation and acclimatisation of the inoculum. The ready biodegradability tests are based on the removal of organic compounds measured as dissolved
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organic carbon (DOC) (OECD 301 A, 301 E, ISO 7827), the production of the catabolic end-product carbon dioxide (e.g. OECD 301 B, ISO 9439, ISO 14593) and the determination of the biochemical oxygen demand (BOD) (e.g. OECD 301 C, 301 D, 301 F, ISO 9408, ISO 10708, ISO 10707). The BOD is usually determined in a respirometer and has the advantage to be a direct biological parameter of aerobic degradation in contrast to DOC removal, which, strictly speaking, indicates the elimination of an organic substance from water, and allows only indirect conclusions about biodegradation to be made. Furthermore, respirometric test systems enable poorly water-soluble compounds to be tested and are very suitable for automated testing. It is therefore not surprising that respirometric test methods play a crucial role in modern biodegradability testing even they have also some disadvantages as for example the high price for the equipment. Improvements to a recently developed respirometric test system were necessary in order to meet all the requirements of precise and accurate test systems which allow the biodegradation testing according to the standardised methods. The authors of this paper were responsible for such improvements and describe in this paper a comparison between a well established test system (Sapromats, . IBUK Konigsbronn, Germany), which has been used for biodegradation studies for decades, and a system (Oxitops, WTW, Weilheim, Germany) which was developed for BOD determinations of wastewater. Emphasis is also laid on the analysis of biodegradation data by modern biomathematical modelling tools to achieve accurate and reliable results from the biodegradation tests.
2. Material and methods 2.1. Chemicals Aniline, benzoic acid, allylthiourea (ATU), trace elements and mineral salts were analytical-grade chemicals purchased from Merck, Darmstadt, Germany. All other chemicals were obtained from BASF, Ludwigshafen, Germany. 2.2. Activated sludge Activated sludge was collected either from a laboratory wastewater treatment plant fed with municipal sewage or from a plant treating predominantly industrial wastewater. The inoculum was preconditioned to reduce the endogenous respiration rate. This was done by sieving the sludge using sieves with 0.8 mm pore size, to remove coarse particles, washing once with tap water, bringing to a concentration of 5 g l1 dry matter and finally aerating for 2 days. This starved sludge suspen-
sion was further diluted to the inoculum concentrations given in the different OECD and ISO protocols.
2.3. Biodegradation tests The methods used were the DOC die-away test (OECD 301A, ISO 7827), the Zahn-Wellens test (OECD 302B, ISO 9888), the CO2 evolution test (OECD 301B, ISO 9439), the CO2 headspace test (ISO 14593) and the manometric respirometry test (OECD 301F, ISO 9408). They were used to determine the ultimate biodegradability or mineralisation of a test compound by the use of the parameters DOC, CO2 and BOD. The mineral medium of the OECD 301, OECD 302 and ISO 14593 test consisted of KH2PO4 (0.625 mM), K2HPO4 (1.249 mM), Na2HPO4*2 H2O (1.877 mM), NH4Cl (0.093 mM), CaCl2 (0.248 mM), MgSO4*7 H2O (0.0913 mM) and FeCl3*6 H2O (0.0009 mM). The concentration of the test compound was 20 mg l1 DOC (OECD 301A, 301B, ISO 14593), 100 mg l1 substance (OECD 301F) and 100 to 400 mg l1 DOC (OECD 302B). The inoculum concentration was 4 mg l1 dry matter (ISO 14593), 30 mg l1 dry matter (OECD 301A, 301B, 301F) and 1000 mg l1 dry matter (OECD 302B). Other important test parameters for these biodegradation tests are given in the respective guidelines. The DOC die-away test was performed in 2-lErlenmeyer flasks filled with 1 l of medium. All flasks were shaken on a rotary shaker at a speed of 150 rpm. Samples were withdrawn at frequent intervals and centrifuged at 4000 rpm for 10 min. The DOC of the supernatant liquid was analysed in a carbon analyser. After subtracting the blanks the measured DOC values were expressed as a percentage of the concentration at the start of the text. The Zahn-Wellens test was performed in 2-litre-flasks equipped with a magnetic stirrer and aerated by diffused air. The handling of DOC samples and the calculation of biodegradation were similar to the DOC die-away test. The degradation of chemical compounds in the CO2 evolution test was determined at a liquid volume of 1.5 l in 2-l cylindrical bottles. CO2-free air was passed through the liquid at a rate of about 1–2 bubbles per second (50 ml min1). Agitation was increased by stirring on a magnetic stirrer. The exhaust gas from the vessels was passed through a series of two bottles each containing 100 ml of 50 mM NaOH to trap the evolved carbon dioxide. The traps were replaced at frequent intervals and the carbon dioxide was determined as total inorganic carbon (TIC) in a carbon analyser (Shimadzu 5000). After subtracting the blank values biodegradation is calculated and expressed as a percentage of the measured carbon dioxide compared to the theoretical carbon dioxide (ThCO2) of the test substance.
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constant temperature during a test run. The decrease in headspace pressure in the closed test vessel is continuously recorded and the biochemical oxygen demand (BOD) is calculated according to the following equation: MðO2 Þ VTotal VLiquid T22 BOD ¼ DpðO2 Þ; þa RT VTotal T0
The CO2 headspace test was performed in 160-ml flasks filled with 100 ml of medium. The sealed vessels (butyl rubber septa and aluminium caps) were placed on an orbital shaker at about 150 rpm to ensure good mixing of the vessel contents. Flasks were sacrificed weekly for analysis by injecting sodium hydroxide to convert the carbon dioxide to carbonate which was determined as TIC. The calculation of biodegradation is similar to the CO2 evolution test. The manometric respirometry tests were carried out in the Sapromat and the Oxitop system [6]. A scheme of the Oxitop and the Sapromat apparatus is shown in Figs. 1A and B respectively. The respirometer equipment and its function are described elsewhere [7,8]. The Sapromat consists of 12 test vessels (nominal volume 500 ml) with carbon dioxide traps in the headspace containing soda lime each connected to a manometer and an electrochemical oxygen production unit. The installation is in a temperature-controlled water bath. If biodegradation takes place in the test mixture (250 ml of inorganic medium, test substance and 30 mg l1 dry matter of activated sludge) oxygen is used by the bacteria and equivalent amounts of carbon dioxide are produced and trapped. A subpressure arises and induces via the manometer the electrochemical production of oxygen until the pressure is even. The flow of the electrical current is measured and used to determine the BOD. BOD values are therefore continuously recorded, transferred to a computer and used to calculate the biodegradation of the test compound as a percentage of its theoretical oxygen demand (ThOD). The Oxitop system offers an individual number of reactors consisting of glass bottles (510 ml nominal volume) with a carbon dioxide trap (sodium hydroxide) in the headspace. The volume of the test mixture is usually 164 ml. The bottles are furnished with a magnetic stirrer and sealed with a cap containing an electronic pressure indicator (Fig. 1A). An incubator box was used to maintain the respirometer units at
where M(O2) is the molecular weight of oxygen (32 g/ mol), R is the gas constant (83.144 mbar/(mol K)), T0 is the temperature at 01C (273.15 K), T22 is the incubation temperature (295.15 K=221C), VTotal is the total volume of the test vessel, VLiquid is the liquid volume in the test vessel, a is the Bunsen absorption coefficient (0.03103) and Dp(O2) is the difference of the partial pressure of oxygen (mbar). Biodegradation is calculated from the measured BOD and the ThOD as in the Sapromat. The major difference between the two respirometers is the supply of oxygen. The Sapromat is able to replace the oxygen which has been used for biodegradation from the electrochemical oxygen production unit. Due to the construction of the test system this replenishing process only occurs if a certain subpressure is reached. In the Oxitop such a replacement of oxygen is not necessary, but care has to be taken that the headspace over the liquid phase in the test vessels is large enough, so that it contains sufficient oxygen and enables complete oxidative biodegradation of the organic test compound. Numerous tests in our laboratory have shown the suitability of this system for the standardised respirometer tests. 2.4. Analytical methods For the respirometric tests it is important to distinguish the oxygen used for carbon oxidation from that for nitrogen oxidation (nitrification). Therefore, the nitrite and nitrate concentration in the test medium were analysed by ion chromatography. The apparatus used were: IC system Dionex DX 300; eluent: 80 mM NaOH
5 3 4
Controller Unit
2
4
3
2
2
Computer
6 5
1 1: magnetic stirrer 2: culture medium 3: CO 2 trap
(A)
4: incubation flask 5: pressure sensor
1: magnetic stirrer 2: incubation flask 3: CO 2 trap
1 4: pressure indicator 5: electrolysis cell 6: culture medium
(B) Fig. 1. Schematic views of respirometers. (A) Oxitop system, (B) Sapromat system.
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in water; pre-column: Dionex AG 10; analytical column: Dionex AS 10; flow rate: 1 ml min1; injection volume: 50 ml; temperature: ambient. The DOC concentrations were analysed with a Shimadzu 5000 carbon analyser.
curve. The following modified logistic function was used [12]:
2.5. Mathematical modelling of biodegradation data
where y is the percent theoretical COD or ThOD, resp. ThCO2, t the time (days), and k the rate constant (days1). L and A are fit variables to be optimised, and the term L=ð1 þ AÞ was necessary to reach the lower extent of degradation of 0 at time t ¼ 0: The span of the function is mainly defined by L: All calculations were performed on a Apple Macintosh computer running KaleidaGraph (Synergy Software, Reading PA, USA) data analysis software.
Biodegradation processes were simulated with two different mathematical models: the first-order plot model and the modified logistic function model. If biodegradation is regarded as a first-order reaction, then the rate of biodegradation is proportional to the concentration of the test chemical [1]: v¼
dS ¼ k1 S; dt
where S is the concentration of the test chemical, t the time (days), k1 the first-order rate constant (days1), and v the rate of test substance disappearance (rate of biodegradation). A transformation of this equation leads to: k1 t
Srem ðtÞ ¼ Sinit e
;
where Srem is the remaining test chemical concentration and Sinit the initial test chemical concentration. Larson and Perry [9], Larson [10] and Srinivasan and Viraraghavan [11] also applied first-order kinetics to determine the rate of oxygen consumption and CO2 formation using the following non-linear fit model: ( 0 for xpc ; y¼ k1 ðtcÞ for x > c að1 e where y is the percent theoretical or chemical oxygen demand, resp. percent CO2 formation (% COD or ThOD resp. % ThCO2), t the time (days), a the asymptote of curve (% COD or ThOD resp. % ThCO2), k1 the first-order rate constant (days1), and c the lag period (days). This model was used for biodegradation curves with a flat lag phase [12]. The theoretical oxygen demand ThOD (mgO2 mg material1) was calculated according to Larson and Perry [9]. The theoretical CO2 formation (ThCO2) was calculated from the carbon content of the test material assuming complete oxidation of the carbon. First-order rate constants can be used to calculate the half-life (t1=2 ) for the test chemical by the equation: t1=2 ¼
ln 2 : k1
The modified logistic model is widely used to describe growth curves for bacteria under substrate limiting conditions. It can also be used for the description of biodegradation processes with an s-shaped or sigmoidal
y¼
L L ; 1 þ Aekt 1 þ A
3. Results 3.1. Endogenous respiration The endogenous BOD is the oxygen consumption resulting from the basic respiration activity of a starved inoculum without any addition of a biodegradable carbon source. In biodegradation tests it is essential to determine this endogenous BOD because the measured BOD data from the test assays with the test substance have to be corrected by these blank values. The extent of endogenous respiration was determined according to the test methods in both respirometer systems. Prior to use the inoculum was starved for about 48 h. After 6, 14 and 28 days of incubation time the BOD values were analysed statistically. After 6 days almost no differences could be observed, but at days 14 and 28 it could be shown with a two-sample t-test that the data from the Oxitop were significantly higher than the data from the Sapromat. The blank BOD data at the end of the test (usually after 28 days) from the Oxitop were about 18–23% higher compared with the data from the Sapromat (Table 1), but they are still in the range for blank values as specified by the standard methods (20– 60 mg l1). The reason for these differences are not yet clear but it could be due to the subsequent delivery of oxygen in the Sapromat, whereas differences of the used activated sludge should play a minor role as the origin of the sludge was the same. Furthermore, time dependent variations of endogenous respiration activity of the sludge could not be detected in both test systems. When testing the influence of different CO2-absorbing materials (NaOH, KOH, sodium lime) on the endogenous respiration activity, no striking differences were found. Therefore we concluded that the absorbing materials had no influence on the endogenous respiration and the differences were mainly due to the different technical design of the two respirometer systems.
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3.2. Different inocula The influence of different inocula on the degradation pattern was investigated in both respirometer systems. Cyclohexanone and 2-ethylhexylacrylate were used as test chemicals. The inocula were activated sludge taken from an industrial wastewater treatment plant and a municipal wastewater treatment plant at a concentration of 30 mg l1 dry matter. The data obtained are summarised in Table 2. For cyclohexanone and 2ethylhexylacrylate only minor differences in the extent of biodegradation were observed but for cyclohexanone a higher rate of degradation could be found with a municipal inoculum. 3.3. Different respirometric test systems
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yacrylate) a higher extent of degradation was observed in the Sapromat using an industrial inoculum. For diethylene glycol a significant higher extent of degradation was found in the Oxitop. The difference of the mean values for NTA was >10% but due to the high standard deviation of the data the mean values did not prove to be significantly different (Students t-test, 95% confidence level). The lag phases in the two different systems were quite consistent, with NTA being the only exception. The k1 constants varied in a certain range but no system showed constantly higher or lower values than the other one. Typically, k1 constants lay in the range of 0.047–0.934 day1, with most data being in a range from 0.10 to 0.30 day1 (Table 3). 3.4. Degradation of aniline in different biodegradation tests
The degradation pattern of ten different chemical compounds was studied in both respirometer systems. Parameters determined were the lag phase, the k1 constants for determining the degradation rate, and the extent of biodegradation, usually expressed as a percentage. The data obtained are summarised in Table 2. In eight cases the extent of degradation was in the same range (difference p10%); in one case (2-ethylhex-
Since aniline is a widely used reference substance for biodegradation tests, its degradation in different OECD tests has been extensively evaluated over many years. The test systems used were the Zahn-Wellens test for comparative purposes (OECD 302 B, ISO 9888), which is a test for inherent biodegradability, the DOC dieaway test (OECD 301 A, ISO 7827), the CO2 evolution
Table 1 BOD data from blank values derived from the Oxitop and Sapromat system. The number of data analyzed was 30 for the Oxitop system and 146 for the Sapromat Time
Test system Oxitop
Day 6 Day 14 Day 28
Sapromat
Mean
St. Dev.
SE Mean
Mean
St. Dev.
SE Mean
11.13 18.60 26.00
4.84 5.77 7.62
1.25 1.49 1.97
10.06 15.34 20.00
3.55 4.69 6.59
0.29 0.40 0.55
St. Dev., standard deviation; SE Mean, standard error of the mean.
Table 2 Influence of different inocula on the degradation of cyclohexanone and ethylhexylacrylate Test compound
Parameter
Oxitop
Sapromat
Municipal sludge
Industrial sludge
Municipal sludge
Industrial sludge
Cyclohexanone
Extent of degradation (%) Lag period (days) k1 -constant (day1)
91 (1.6) 0.5 (0.1) 0.224 (0.006)
91 (1.0) 0.5 (0.1) 0.126 (0.005)
96 (6.7) 0.5 (0.1) 0.216 (0.065)
83 (12.2) 0.5 (0.1) 0.162 (0.019)
2-Ethylhexylacrylate
Extent of degradation (%) Lag period (days) k1 -constant (day1)
58 (7.6) 1 (0.1) 0.047 (0.008)
56 (2.1) 1 (0.2) 0.058 (0.003)
73 (7.5) 1 (0.2) 0.122 (0.012)
71 (3.1) 1 (0.2) 0.106 (0.013)
The standard deviation of the results is given in brackets. Tests were performed in triplicate.
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3.5. Nitrification processes and their inhibition
test (OECD 301 B, ISO 9439) and the manometric respirometry test (OECD 301 F, ISO 9408). Again the Sapromat and Oxitop systems were both used in parallel. The data summarised in Table 4 show that the k1 constants are comparable in both respirometric systems, whereas the k1 constants in the DOC die-away test are the highest and in the CO2 evolution test, the lowest. The lag periods are comparable in the Oxitop system, the DOC die-away test and the CO2 evolution test, but longer in the Sapromat test system. Also, the final extent of degradation is comparable in the Oxitop, the DOC die-away test and the CO2 evolution test, whereas in the Sapromat a lower extent of degradation is observed. When testing aniline degradation in the Oxitop with different carbon dioxide absorbing materials (NaOH, KOH and sodium lime) it was found that similar degradation curves were obtained.
Nitrification processes have a striking effect on the biochemical oxygen demand, because 1 g of organic nitrogen released as ammonium is equivalent to 4.57 g of oxygen. In general, the ammonium sources in biodegradation tests are the mineral medium and the inoculum, but depending on the test conditions and the test duration also nitrogenous organic compounds can contribute to the nitrification and hence influence the test results of respirometric tests. This can be compensated for by either subtracting the oxygen in the resultant nitrate and nitrite from the measured BOD values as it is foreseen in the standard method or by preventing nitrification by the addition of a specific inhibitor such as allylthiourea (ATU). As can be seen in Figs. 2 and 3 a concentration of 10 mg l1 ATU
Table 3 Degradation of different chemical compounds in respirometric test systems Test compound
Test system Sapromat
Acrylic acid Benzoic acid Diethylene glycol Glycerol Morpholine NTA 1.5-Pentanediol Phenol
Oxitop
Degradation (%)
k1 -constant (day1)
Lag period (days)
Degradation (%)
k1 -constant (day1)
Lag period (days)
94 91 59 68 87 81 83 81
0.934 0.556 0.081 0.258 0.120 0.214 0.342 0.224
2.5 (0.3) 0 (0.1) 5 (1.0) 1 (0.25) 16 (2.2) 14 (2.08) 3 (0.45) 3 (0.45)
91 93 89 78 89 70 89 76
0.993 0.461 0.173 0.200 0.248 0.564 0.222 0.412
3 (0.2) 0 (0.1) 5 (1.0) 1 (1.0) 16 (1.8) 9 (1.0) 3 (0.3) 3 (0.3)
(3.78) (9.09) (12.03) (5.29) (5.77) (11.06) (8.51) (2.52)
(0.29) (0.17) (0.026) (0.083) (0.038) (0.064) (0.11) (0.06)
(12.06) (0.83) (3.63) (2.89) (5.77) (10.23) (6.11) (12.19)
(0.32) (0.14) (0.04) (0.04) (0.06) (0.12) (0.05) (0.10)
The number of replicate tests performed was 3 (acrylic acid, glycerol, morpholine, 1.5-pentanediol, phenol) to 6 (benzoic acid, diethylene glycol, NTA). The standard deviation of the results is given in brackets.
Table 4 Degradation of aniline in different OECD biodegradation tests and in the CO2 headspace test Test system
Parameter k1 -constant (day1)
Lag period (days)
Extent of degradation (%)
Number of tests performed
OECD 301 DOC-die-away test OECD 301 B Sturm test OECD 301 F Oxitop OECD 301 F Sapromat CO2-headspace test
1.71 0.30 0.74 0.86 n.d.
2.2 (0.94) 3.0 (0.71) 3.6 (0.8) 6.0 (2.1) n.d.
91 51 13 150 22
OECD 302 B Zahn-Wellens test
0.096
96 87 92 81 87 97 98
a
(0.55) (0.12) (0.25) (0.21)
CO2 evolution. DOC elimination. n.d. not determined. The standard deviation of the results is given in brackets. b
3
(3.0) (9.0) (12.8) (11.4) (6.0)a (3.0)b
1
P. Reuschenbach et al. / Water Research 37 (2003) 1571–1582
1.6
1.6
without ATU with ATU
1.2 1 0.8 0.6 0.4 0.2
(A)
without ATU with ATU
1.4 Degradation (BOD/ThOD)
Degradation (BOD/ThOD)
1.4
0
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1.2 1 0.8 0.6 0.4 0.2
0
5
10
15 20 Time (days)
25
0
30 (B)
0
5
10
15 20 Time (days)
25
30
Fig. 2. Influence of allylthiourea (ATU) on the oxygen demand during the degradation of aniline (A) and isopropylamine (B) in the Oxitop system.
Fig. 3. Influence of allylthiourea (ATU) on the oxygen demand during the degradation of aniline (A) and isopropylamine (B) in the Sapromat system.
effectively inhibits nitrification. At this concentration the heterotrophic respiration is not effected. Nitrification processes occur in most OECD 301 tests, the extent differs, however. Aniline, for example, was usually not nitrified in the Sapromat whereas it was nitrified in the Oxitop without addition of ATU. The measured concentrations of nitrite and nitrate ranged from about 0.02 to 0.9 mM after an incubation period of 28 days (Fig. 4). The reason for the missing nitrification still remains unclear. For all other test conditions were identical, it can only be due to the different construction of the respirometers. The isopropylamin nitrogen, on the other hand, was nitrified in both respirometer systems.
3.6. Biodegradation modelling Different mathematical approaches were used to model the biodegradation kinetics data obtained. The models used were a modified logistic plot and a firstorder plot. An example for the biodegradation of morpholine and diethylene glycol is shown in Figs. 5A–D and 6A–D. It is obvious that the modified logistic plot can be used for all sigmoidal biodegradation curves, whereas the first-order plot is suitable for biodegradation curves with flat lag periods. On the other hand, the first-order plot is a tool that can be used to determine easily the half-time constant of a chemical compound. The prerequisite for the use of a first-order
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Fig. 4. Nitrification in different biodegradation test systems. The formation of nitrate/nitrite-N is illustrated in blank controls (A) and degradation assays containing aniline (B). The mean values of 7 to 14 investigations are shown. The test systems used are (1) DOC dieaway test (OECD 301 A), (2) CO2 evolution test (OECD 301 B), (3) manomeric respirometry test (Sapromat, OECD 301 F), (4) manomeric respirometry test (Oxitop, OECD 301 F), (5) CO2-headspace test.
plot is that there are enough data in the exponential phase (in general to 6 8 data points) to yield sufficient accuracy.
4. Discussion According to OECD guidelines and ISO standards blank values for endogenous respiration should be in the range of 20–30 mg l1 and not exceed 60 mg l1. Otherwise the experimental technique has to be examined or the test repeated with another inoculum [3] The data reported in this study are in the given range for both respirometric systems. Endogenous respiration processes are due to aerobic biodegradation of residual carbon sources of the inoculum and self-digestion processes present. A range of 20–30 mg l1 BOD in the blank is equivalent to a ThOD of 7.5 to 11.25 mg l1 of carbon in glucose (0.6–0.9 mM of carbon). When comparing the ThOD or the chemical oxygen demand (COD) added to the test system at the recommended test concentrations (100 mg l1 substance or 50–100 mg l1 ThOD), the endogenous respiration represents a fraction of 0.2–0.6 of the total oxygen demand and is therefore significant. The most convenient way to reduce endogenous respiration is to starve the inoculum. The drawback of this method is the possible reduction in the number of viable bacterial cells and subsequently the loss of biodegradation potential. A comparison of the two different respirometric test systems with numerous test substances revealed that both systems can give different data. The advantage of the Oxitop is its compact design, the accuracy and
reliability of the measuring units, the easy reading of the measured data and its transfer to a computer and a relatively low price. The main advantage of the Sapromat is the oxygen replacement and the option possibility to extend the duration nearly infinitely. Another important point in biodegradation tests is the source of the inoculum. It is known that inocula from industrial wastewater treatment plants show generally a higher biodegradation potential for chemical compounds than inocula from municipal wastewater treatment plants. In this study, we were, however, unable to confirm this assumption. For example, the degradation rate of cyclohexanone was faster with municipal activated sludge than with industrial activated sludge. On the other hand, with 2-ethylhexylacrylate, inocula from both sources showed comparable biodegradation rates. In the case of morpholine, lag phases of 15–16 days were observed before the onset of biodegradation processes. This extended adaptation period has also been reported in former studies [13]. Therefore, it is possible that, due to the extended lag phase and the low inoculum concentrations in OECD 301 tests, the number of bacteria may not always be sufficient for immediate biodegradation. However, a mixture of different inocula may deliver the sufficient diversity and the use of a degradable substrate in addition to the test substance may enable cometabolism to eventually bring about complete biodegradation of recalcitrant test substances. These facts underline the stringency of the OECD 301 tests. This stringency of the test conditions together with alterations in the metabolic activity of the inoculum used may also be the reason for the variations in biodegradability as observed in both respirometric test systems. By
P. Reuschenbach et al. / Water Research 37 (2003) 1571–1582
Oxitop (k1=0.248 day-1)
Oxitop (k = 0.76 day-1) Sapromat (k = 0.46 day-1)
Sapromat (k1=0.120 day-1) 1
1
0.8
0.8 Degradation
Degradation
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0.6 0.4
0.6 0.4 0.2
0.2
0
0 15
20 25 Time (days)
(A)
0
30
5
10
15
20
25
30
Time (days)
(B)
Oxitop (k = 0.43 day-1) Sapromat (k = 0.15 day-1)
Oxitop (k1=0.173 day-1) Sapromat (k1=0.081 day-1)
1
0.9
0.8 Degradation
0.8
Degradation
0.7 0.6 0.5 0.4
0.6 0.4
0.3
0.2
0.2
0
0.1 5
(C)
10
15
20
25
0
30
Time (days)
(D)
5
10
15
20
25
30
Time (days)
Fig. 5. Kinetical analysis of the degradation of morpholine (A,B) and diethylene glycol (C,D). The data were analysed with first-order plots (A, C) and a modified logistic function (B,D).
parallel testing with a recommended control substance (aniline) major alterations in the quality of the inoculum used are detected. The suboptimal test conditions of the OECD 301 tests especially for adaption processes have also been recognised by the OECD, but are regarded as necessary for a realistic simulation of biodegradation in surface water. In this way variations of the test results may reflect variations in biodegradation pattern under realistic environmental conditions. In our investigations we found that the k1 constants for aniline were highest in the DOC die-away test and lowest in the CO2 evolution test (Table 4). This observation is of general interest for the determination of ready biodegradability according to the OECD criteria and may be explained as follows. The DOC removal from water by adsorption on the biomass or by uptake into the cells is the first step and has therefore the
highest rate. The consumption of oxygen for oxidative processes and finally the production of carbon dioxide are subsequent steps with lower rates. DOC removal and the depletion of oxygen in the medium can be monitored directly in the test vessel, whereas the carbon dioxide is measured in the traps outside the test medium. Due to the necessary liberation of carbon dioxide from the liquid phase in the test vessel and the subsequent diffusion processes into the trap, lower biodegradation is reasonable for this type of test. A disadvantage of test systems based on BOD and CO2 is the difficulty to differentiate between the oxidised part of the test substance and the part incorporated in biomass which can be expressed by the heterotrophic yield coefficient [14,15]. Heterotrophic yield coefficients can vary between 0.40 and 0.80 and are dependent from the substrate used, the source of the inoculum, the ratio
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municipal inoculum (k = 0.63 day-1) industrial inoculum (k = 0.24 day-1)
municipal inoculum (k = 0.32 day-1) industrial inoculum (k = 0.18 day-1)
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Fig. 6. Degradation of cyclohexanone in the Oxitop (A, C) and Sapromat system (B, D). Municipal and industrial inocula were used for these biodegradation studies. The data were analysed with first-order plots (C,D) and a modified logistic function (A, B).
of substrate to inoculum and the test conditions used. Therefore, higher extents of degradation in tests based on carbon removal in comparison to tests based on oxygen consumption and carbon dioxide formation can be explained in this way. In general, extents of biodegradation of >60% (BOD of THOD or CO2 of THCO2) indicate a sufficient biodegradation [3]. Test results based on DOC removal, on the other hand, seem to be much more reliable as degrees of >90% indicate good degradation. This discrepancy could be solved either by performing expensive carbon balances, which allow a clear understanding of the fate of the test substance or by the use of limit values and degradation criteria. Experts have defined the OECD criteria for ready biodegradability and if they are fulfilled the test substance is from definition biodegradable. The biodegradation patterns or curves of standardised batch tests are described by the lag phase, the final extent of biodegradation in the plateau phase and the time taken to reach the plateau. A modern approach to describing
biodegradation kinetics is the use of mathematical models. A variety of such methods exist which can be applied [1]. In this study we focussed on two principal procedures. In first-order reactions the rate of biodegradation is only proportional to the concentration of the test substance. Linear regression procedures are usually used to determine the first-order rate constant k1 ; but it is also possible to use non-linear procedures [10,11,16,17]. In the literature, a number of k1 constants have been published for different industrial chemicals such as benzene [18], chlorobenzene [19], 4-chlorophenol [18], methyl parathion [20], nitrilotriacetic acid (NTA) [17,21,22]. The first-order rate constants range from 0.01 to 4.8 day1. Most of the differences reported were due to the quantity and quality of the biomass used [1]. To calculate first-order kinetics the determination of sufficient biodegradation data in the degradation phase is necessary. In order to model the biodegradation curve from the lag phase to the plateau phase, the modified logistic model was used. This mathematical model is
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based on a logistic function, which is often used to describe the population pattern under growth limiting conditions [23]. Other methods frequently used are the Monod growth model with and without growth of biomass, the zero order model and the logarithmic model. A critical discussion of these models was published by Simkins and Alexander [24]. For future standards it could be advantageous to integrate these modern mathematical tools into standardised methods. For example, the first-order kinetic model could be used instead of the 10-day-window to describe degradation kinetics, and the modified logistic model could be appropriate in estimating the lag phase as well as the final extent of degradation. There are already ISO standards available which are used to determine biodegradation kinetics (ISO 14592 parts 1 and 2) indicating the positive future perspectives of biodegradation kinetics.
5. Conclusions The Oxitop and Sapromat method were found to be reliable respirometric test methods for assessing the biodegradability of chemical compounds. In the Oxitop system endogenous BOD production proved to be significantly higher after 14 and 28 days of incubation. On the other hand after 6 days of incubation the differences were not significant. When testing municipal and industrial activated sludge as inocula minor differences in the extent of biodegradation could be stated for cyclohexanone and 2-ethylhexylacrylate. On the other hand, the degradation rate of cyclohexanone was higher with a municipal inoulum. Both respirometric systems were used to evaluate the biodegradability of different chemical compounds. In eight cases comparable results were found. In two cases (diethylene glycol and 2-ethylhexylacrylate) different results were obtained indicating that the test systems are not always equivalent. Aniline is a recommended reference compound for biodegradation tests. In different OECD and ISO test systems it was found that in the Sapromat longer lag periods and a lower extent of biodegradation occurred. As expected the degradation rate constant k1 was highest in the DOC die-away test (OECD 301A) and lowest in the CO2 evolution test (OECD 301B). Allylthiourea (ATU) at a final concentration of 10 mg l1 can effectively inhibit nitrification processes which may occur during biodegradation studies. At this concentration the heterotrophic degradation activity is not disturbed. In nearly all OECD and ISO test systems nitrification processes were observed. Only when testing aniline in the Sapromat system no nitrification could be found.
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Biodegradation modelling could be performed by using a first-order plot for biodegradation curves with a flat lag period and a modified logistic plot for sigmoidal biodegradation curves.
References [1] Battersby NS. A review of biodegradation kinetics in the aquatic environment. Chemosphere 1990;21:1243–84. [2] Swisher RD. Surfactant biodegradation, 2nd ed. New York: Marcel Dekker Inc., 1987. [3] OECD Guidelines. OECD Guidelines for testing of chemicals, Paris, 1993. OECD 301 A: DOC Die-AwayTest; OECD 301 B: CO2 Evolution Test; OECD 301 C: Modified MITI Test (I); OECD 301 D: Closed Bottle Test; OECD 301 E: Modified OECD Screening Test; OECD 301 F: Manometric Respirometry Test; OECD 302 B: ZahnWellens/EMPA test. [4] ISO (International Organization for Standardization). ISO 7827 (1994): Water Quality—Evaluation in an aqueous medium of the ‘‘ultimate’’ aerobic biodegradability of organic compounds—method by analysis of dissolved organic carbon; ISO 9439 (1999): Water Quality—Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium—Carbon dioxide evolution test; ISO 9408 (1999): Water Quality—Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium by determination of oxygen demand in a closed respirometer; ISO 9888 (1999): Water Quality— Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium—Static test (Zahn-Wellens method); ISO 10707 (1994): Water Quality—Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium—method by analysis of biochemical oxygen demand (closed bottle test); ISO 10708 (1997): Water Quality—Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium—method by determining the biochemical oxygen demand in a two-phase closed bottle test; ISO 14592 (draft): Water Quality—Evaluation of the aerobic biodegradability of organic compounds at low concentrations— Part 1: Shake flask batch test with surface water or surface water sediment suspensions—Part 2: Continuous river flow model with attached biomass; ISO 14593 (1999): Water Quality—Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium—Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test); ISO 15462 (1997): Water Quality— Selection of tests for biodegradability (technical report). [5] Pagga U. Testing biodegradability with standardized methods. Chemosphere 1997;35:2953–72. [6] Conzelmann F, Beitlich R. Einfache Bestimmung der biologischen Abbaubarkeit nach OECD 301 F mit dem Oxitop (A simple determination method of biodegradability according to OECD 301 F with the Oxitop system). Melliand, October 1996. p. 700. [7] Govind R, Gao C, Lai L, Tabak HH. Continuous, automated and simultaneous measurement of oxygen uptake and carbon dioxide evolution in biological systems. Water Envrion Res 1997;69(1):73.
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[8] Pagga U. Respirometric degradation and toxicity test with activated sludge for substances and waste water. Vom Wasser 1980;55(3):13–326. [9] Larson RJ, Perry RL. Use of the electrolytic respirometer to measure biodegradation in natural waters. Water Res 1981;15:697–702. [10] Larson RJ. Estimation of biodegradation potential of xenobiotic organic chemicals. Appl Environ Microbiol 1979;38:1153–61. [11] Srinivasan PT, Viraraghavan T. An analysis of the ‘‘Modified Sturm test’’ data. Chemosphere 2000;40:99–102. [12] Larson RJ, Hansmann MA, Bookland EA. Carbon dioxide recovery in ready biodegradation tests: mass transfer and kinetic considerations. Chemosphere 1996;33:1195–210. [13] Strotmann UJ, Weberru U, Bias WR. Biodegradation of morpholine in several biodegradation tests and in wastewater treatment plants. Chemosphere 1993;26:1729–42. [14] Strotmann UJ, Geldern A, Kuhn A, Gendig C, Klein S. Evaluation of a respirometric test method to determine the heterotrophic yield coefficient of activated sludge bacteria. Chemosphere 1999;38:3555–70. [15] Kappeler J, Gujer W. Estimation of kinetic parameters of heterotrophic biomass under aerobic conditions and characterization of wastewater for activated sludge modelling. Water Sci Technol 1992;25:125–39. [16] Larson RJ. Role of biodegradation kinetics in predicting environmental fate. In: Maki AW, Dickson KL, Cairns J, editors. Biotransformation and fate of chemicals in the aquatic environment. Washington: American Society for Microbiology, 1980. p. 67–86.
[17] Larson RJ. Kinetic and ecological approaches for predicting biodegradation rates of organic chemicals in natural ecosystems. In: Klug MJ, Reddy CA, editors. Current perspectives in microbial ecology. Washington: American Society for Microbiology, 1984. p. 677–86. [18] Lee RF, Ryan C. Microbial degradation of organochlorine compounds in estuarine waters and sediments. In: Bourquin AW, Pritchard PH, editors. Proceedings of the Workshop Microbial Degradation of Pollutants in Marine Environments: EPA 600/9-79-012, 1979. p. 443–50. [19] Bartholomev GW, Pfaender FK. Influence of spatial and temporal variations on organic pollutant biodegradation rates in an estuarine environment. Appl Environ Microbiol 1983;45:103–9. [20] Cripe CR, Walker WW, Pritchard PH, Bourqui AW. A shake-flask test for estimation of bodegrdability of toxic organice substances in the aquatic environment. Ecotoxicol Environ Saf 1987;14:239–51. [21] Larson RJ. Comparison of biodegradation rates in laboratory screening studies with rates in natural waters. Residue Rev 1983;85:159–71. [22] Larson RJ, Ventullo RM. Kinetics of biodegradation of nitrilotriacetic acid (NTA) in estuarine environment. Ecotoxicol Environ Saf 1986;12:166–79. [23] Finney RL, Weir MD, Giordano FR. Thomas’ calculus, 10th ed. Boston, San Francisco, New York: Addison Wesley, Longman, 2001. p. 514–518. [24] Simkins S, Alexander M. Models for mineralisation kinetics with variables of substrate concentration and population density. Appl Environ Microbiol 1984;47: 1299–306.
A critical comparison of respirometric biodegradation tests based on OECD 301 and related test methods Peter Reuschenbacha, Udo Paggaa, Uwe Strotmannb,* b
a BASF Aktiengesellschaft, Laboratory of Ecological Studies GV/TC Z570, D-67056 Ludwigshafen, Germany Department of Environmental Technology, Fachhochschule Gelsenkirchen, University of Applied Sciences, Neidenburger Str. 10, D-45877 Gelsenkirchen, Germany
Received 8 January 2002; received in revised form 14 June 2002; accepted 22 October 2002
Abstract Biodegradation studies of organic compounds in the aquatic environment gain important information for the final fate of chemicals in the environment. A decisive role play tests for ready biodegradability (OECD 301) and in this context, the respirometric test (OECD 301F). Two different respirometric systems (Oxitops and Sapromats) were compared and in two of ten cases (diethylene glycol and 2-ethylhexylacrylate) differences were observed indicating that the test systems are not always equivalent. For 2-ethylhexylacrylate and cyclohexanone we could not state differences in the extent of biodegradation with a municipal and industrial inoculum whereas for cyclohexanone the degradation rate was faster with a municipal inoculum. Allylthiourea (ATU) proved to be an effective inhibitor of nitrification processes and did not affect the heterotrophic biodegradation activity. Modelling of biodegradation processes could be successfully performed with a first-order and a modified logistic plot. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Biodegradation; Mathematical modelling; OECD 301 tests; Ready biodegradability; Respirometry
1. Introduction Over past 40 years, the fate of chemical pollutions in the environment has become an important issue. Methods for investigating and monitoring biodegradation processes have been developed [1]. Most efforts have concentrated on the fate of chemicals in the aquatic environment, especially in wastewater treatment processes. Although the elimination of chemicals from the aquatic environment may occur by abiotic processes such as adsorption, hydrolysis and photolysis, the complete conversion of organic chemicals to inorganic products is due to microbial biodegradation processes. During these ultimate biodegradation processes the *Corresponding author. Tel.: +49-209-9596-142; fax: +49209-9596-144. E-mail address: [email protected] (U. Strotmann).
organic matter is converted into CO2, H2O, inorganic salts, microbial biomass and organic metabolites [2]. Nearly 20 years ago, in 1981 the OECD first published its guidelines for testing the biodegradation of chemicals which were updated 1993 [3]. In the mean time, also a number of biodegradation standards of the International Organisation of Standardisation (ISO) [4], which are to some extent similar to the OECD guidelines, have been created. An overview gives ISO 15462 and Pagga [5]. In the OECD guidelines biodegradation tests are divided into three principal categories: tests for ready biodegradability, tests for inherent biodegradability and simulation tests. The most important tests for practical use are the tests for ready biodegradability. These are the most stringent tests, offering only limited opportunities for biodegradation and acclimatisation of the inoculum. The ready biodegradability tests are based on the removal of organic compounds measured as dissolved
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organic carbon (DOC) (OECD 301 A, 301 E, ISO 7827), the production of the catabolic end-product carbon dioxide (e.g. OECD 301 B, ISO 9439, ISO 14593) and the determination of the biochemical oxygen demand (BOD) (e.g. OECD 301 C, 301 D, 301 F, ISO 9408, ISO 10708, ISO 10707). The BOD is usually determined in a respirometer and has the advantage to be a direct biological parameter of aerobic degradation in contrast to DOC removal, which, strictly speaking, indicates the elimination of an organic substance from water, and allows only indirect conclusions about biodegradation to be made. Furthermore, respirometric test systems enable poorly water-soluble compounds to be tested and are very suitable for automated testing. It is therefore not surprising that respirometric test methods play a crucial role in modern biodegradability testing even they have also some disadvantages as for example the high price for the equipment. Improvements to a recently developed respirometric test system were necessary in order to meet all the requirements of precise and accurate test systems which allow the biodegradation testing according to the standardised methods. The authors of this paper were responsible for such improvements and describe in this paper a comparison between a well established test system (Sapromats, . IBUK Konigsbronn, Germany), which has been used for biodegradation studies for decades, and a system (Oxitops, WTW, Weilheim, Germany) which was developed for BOD determinations of wastewater. Emphasis is also laid on the analysis of biodegradation data by modern biomathematical modelling tools to achieve accurate and reliable results from the biodegradation tests.
2. Material and methods 2.1. Chemicals Aniline, benzoic acid, allylthiourea (ATU), trace elements and mineral salts were analytical-grade chemicals purchased from Merck, Darmstadt, Germany. All other chemicals were obtained from BASF, Ludwigshafen, Germany. 2.2. Activated sludge Activated sludge was collected either from a laboratory wastewater treatment plant fed with municipal sewage or from a plant treating predominantly industrial wastewater. The inoculum was preconditioned to reduce the endogenous respiration rate. This was done by sieving the sludge using sieves with 0.8 mm pore size, to remove coarse particles, washing once with tap water, bringing to a concentration of 5 g l1 dry matter and finally aerating for 2 days. This starved sludge suspen-
sion was further diluted to the inoculum concentrations given in the different OECD and ISO protocols.
2.3. Biodegradation tests The methods used were the DOC die-away test (OECD 301A, ISO 7827), the Zahn-Wellens test (OECD 302B, ISO 9888), the CO2 evolution test (OECD 301B, ISO 9439), the CO2 headspace test (ISO 14593) and the manometric respirometry test (OECD 301F, ISO 9408). They were used to determine the ultimate biodegradability or mineralisation of a test compound by the use of the parameters DOC, CO2 and BOD. The mineral medium of the OECD 301, OECD 302 and ISO 14593 test consisted of KH2PO4 (0.625 mM), K2HPO4 (1.249 mM), Na2HPO4*2 H2O (1.877 mM), NH4Cl (0.093 mM), CaCl2 (0.248 mM), MgSO4*7 H2O (0.0913 mM) and FeCl3*6 H2O (0.0009 mM). The concentration of the test compound was 20 mg l1 DOC (OECD 301A, 301B, ISO 14593), 100 mg l1 substance (OECD 301F) and 100 to 400 mg l1 DOC (OECD 302B). The inoculum concentration was 4 mg l1 dry matter (ISO 14593), 30 mg l1 dry matter (OECD 301A, 301B, 301F) and 1000 mg l1 dry matter (OECD 302B). Other important test parameters for these biodegradation tests are given in the respective guidelines. The DOC die-away test was performed in 2-lErlenmeyer flasks filled with 1 l of medium. All flasks were shaken on a rotary shaker at a speed of 150 rpm. Samples were withdrawn at frequent intervals and centrifuged at 4000 rpm for 10 min. The DOC of the supernatant liquid was analysed in a carbon analyser. After subtracting the blanks the measured DOC values were expressed as a percentage of the concentration at the start of the text. The Zahn-Wellens test was performed in 2-litre-flasks equipped with a magnetic stirrer and aerated by diffused air. The handling of DOC samples and the calculation of biodegradation were similar to the DOC die-away test. The degradation of chemical compounds in the CO2 evolution test was determined at a liquid volume of 1.5 l in 2-l cylindrical bottles. CO2-free air was passed through the liquid at a rate of about 1–2 bubbles per second (50 ml min1). Agitation was increased by stirring on a magnetic stirrer. The exhaust gas from the vessels was passed through a series of two bottles each containing 100 ml of 50 mM NaOH to trap the evolved carbon dioxide. The traps were replaced at frequent intervals and the carbon dioxide was determined as total inorganic carbon (TIC) in a carbon analyser (Shimadzu 5000). After subtracting the blank values biodegradation is calculated and expressed as a percentage of the measured carbon dioxide compared to the theoretical carbon dioxide (ThCO2) of the test substance.
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constant temperature during a test run. The decrease in headspace pressure in the closed test vessel is continuously recorded and the biochemical oxygen demand (BOD) is calculated according to the following equation: MðO2 Þ VTotal VLiquid T22 BOD ¼ DpðO2 Þ; þa RT VTotal T0
The CO2 headspace test was performed in 160-ml flasks filled with 100 ml of medium. The sealed vessels (butyl rubber septa and aluminium caps) were placed on an orbital shaker at about 150 rpm to ensure good mixing of the vessel contents. Flasks were sacrificed weekly for analysis by injecting sodium hydroxide to convert the carbon dioxide to carbonate which was determined as TIC. The calculation of biodegradation is similar to the CO2 evolution test. The manometric respirometry tests were carried out in the Sapromat and the Oxitop system [6]. A scheme of the Oxitop and the Sapromat apparatus is shown in Figs. 1A and B respectively. The respirometer equipment and its function are described elsewhere [7,8]. The Sapromat consists of 12 test vessels (nominal volume 500 ml) with carbon dioxide traps in the headspace containing soda lime each connected to a manometer and an electrochemical oxygen production unit. The installation is in a temperature-controlled water bath. If biodegradation takes place in the test mixture (250 ml of inorganic medium, test substance and 30 mg l1 dry matter of activated sludge) oxygen is used by the bacteria and equivalent amounts of carbon dioxide are produced and trapped. A subpressure arises and induces via the manometer the electrochemical production of oxygen until the pressure is even. The flow of the electrical current is measured and used to determine the BOD. BOD values are therefore continuously recorded, transferred to a computer and used to calculate the biodegradation of the test compound as a percentage of its theoretical oxygen demand (ThOD). The Oxitop system offers an individual number of reactors consisting of glass bottles (510 ml nominal volume) with a carbon dioxide trap (sodium hydroxide) in the headspace. The volume of the test mixture is usually 164 ml. The bottles are furnished with a magnetic stirrer and sealed with a cap containing an electronic pressure indicator (Fig. 1A). An incubator box was used to maintain the respirometer units at
where M(O2) is the molecular weight of oxygen (32 g/ mol), R is the gas constant (83.144 mbar/(mol K)), T0 is the temperature at 01C (273.15 K), T22 is the incubation temperature (295.15 K=221C), VTotal is the total volume of the test vessel, VLiquid is the liquid volume in the test vessel, a is the Bunsen absorption coefficient (0.03103) and Dp(O2) is the difference of the partial pressure of oxygen (mbar). Biodegradation is calculated from the measured BOD and the ThOD as in the Sapromat. The major difference between the two respirometers is the supply of oxygen. The Sapromat is able to replace the oxygen which has been used for biodegradation from the electrochemical oxygen production unit. Due to the construction of the test system this replenishing process only occurs if a certain subpressure is reached. In the Oxitop such a replacement of oxygen is not necessary, but care has to be taken that the headspace over the liquid phase in the test vessels is large enough, so that it contains sufficient oxygen and enables complete oxidative biodegradation of the organic test compound. Numerous tests in our laboratory have shown the suitability of this system for the standardised respirometer tests. 2.4. Analytical methods For the respirometric tests it is important to distinguish the oxygen used for carbon oxidation from that for nitrogen oxidation (nitrification). Therefore, the nitrite and nitrate concentration in the test medium were analysed by ion chromatography. The apparatus used were: IC system Dionex DX 300; eluent: 80 mM NaOH
5 3 4
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4: incubation flask 5: pressure sensor
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(B) Fig. 1. Schematic views of respirometers. (A) Oxitop system, (B) Sapromat system.
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in water; pre-column: Dionex AG 10; analytical column: Dionex AS 10; flow rate: 1 ml min1; injection volume: 50 ml; temperature: ambient. The DOC concentrations were analysed with a Shimadzu 5000 carbon analyser.
curve. The following modified logistic function was used [12]:
2.5. Mathematical modelling of biodegradation data
where y is the percent theoretical COD or ThOD, resp. ThCO2, t the time (days), and k the rate constant (days1). L and A are fit variables to be optimised, and the term L=ð1 þ AÞ was necessary to reach the lower extent of degradation of 0 at time t ¼ 0: The span of the function is mainly defined by L: All calculations were performed on a Apple Macintosh computer running KaleidaGraph (Synergy Software, Reading PA, USA) data analysis software.
Biodegradation processes were simulated with two different mathematical models: the first-order plot model and the modified logistic function model. If biodegradation is regarded as a first-order reaction, then the rate of biodegradation is proportional to the concentration of the test chemical [1]: v¼
dS ¼ k1 S; dt
where S is the concentration of the test chemical, t the time (days), k1 the first-order rate constant (days1), and v the rate of test substance disappearance (rate of biodegradation). A transformation of this equation leads to: k1 t
Srem ðtÞ ¼ Sinit e
;
where Srem is the remaining test chemical concentration and Sinit the initial test chemical concentration. Larson and Perry [9], Larson [10] and Srinivasan and Viraraghavan [11] also applied first-order kinetics to determine the rate of oxygen consumption and CO2 formation using the following non-linear fit model: ( 0 for xpc ; y¼ k1 ðtcÞ for x > c að1 e where y is the percent theoretical or chemical oxygen demand, resp. percent CO2 formation (% COD or ThOD resp. % ThCO2), t the time (days), a the asymptote of curve (% COD or ThOD resp. % ThCO2), k1 the first-order rate constant (days1), and c the lag period (days). This model was used for biodegradation curves with a flat lag phase [12]. The theoretical oxygen demand ThOD (mgO2 mg material1) was calculated according to Larson and Perry [9]. The theoretical CO2 formation (ThCO2) was calculated from the carbon content of the test material assuming complete oxidation of the carbon. First-order rate constants can be used to calculate the half-life (t1=2 ) for the test chemical by the equation: t1=2 ¼
ln 2 : k1
The modified logistic model is widely used to describe growth curves for bacteria under substrate limiting conditions. It can also be used for the description of biodegradation processes with an s-shaped or sigmoidal
y¼
L L ; 1 þ Aekt 1 þ A
3. Results 3.1. Endogenous respiration The endogenous BOD is the oxygen consumption resulting from the basic respiration activity of a starved inoculum without any addition of a biodegradable carbon source. In biodegradation tests it is essential to determine this endogenous BOD because the measured BOD data from the test assays with the test substance have to be corrected by these blank values. The extent of endogenous respiration was determined according to the test methods in both respirometer systems. Prior to use the inoculum was starved for about 48 h. After 6, 14 and 28 days of incubation time the BOD values were analysed statistically. After 6 days almost no differences could be observed, but at days 14 and 28 it could be shown with a two-sample t-test that the data from the Oxitop were significantly higher than the data from the Sapromat. The blank BOD data at the end of the test (usually after 28 days) from the Oxitop were about 18–23% higher compared with the data from the Sapromat (Table 1), but they are still in the range for blank values as specified by the standard methods (20– 60 mg l1). The reason for these differences are not yet clear but it could be due to the subsequent delivery of oxygen in the Sapromat, whereas differences of the used activated sludge should play a minor role as the origin of the sludge was the same. Furthermore, time dependent variations of endogenous respiration activity of the sludge could not be detected in both test systems. When testing the influence of different CO2-absorbing materials (NaOH, KOH, sodium lime) on the endogenous respiration activity, no striking differences were found. Therefore we concluded that the absorbing materials had no influence on the endogenous respiration and the differences were mainly due to the different technical design of the two respirometer systems.
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3.2. Different inocula The influence of different inocula on the degradation pattern was investigated in both respirometer systems. Cyclohexanone and 2-ethylhexylacrylate were used as test chemicals. The inocula were activated sludge taken from an industrial wastewater treatment plant and a municipal wastewater treatment plant at a concentration of 30 mg l1 dry matter. The data obtained are summarised in Table 2. For cyclohexanone and 2ethylhexylacrylate only minor differences in the extent of biodegradation were observed but for cyclohexanone a higher rate of degradation could be found with a municipal inoculum. 3.3. Different respirometric test systems
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yacrylate) a higher extent of degradation was observed in the Sapromat using an industrial inoculum. For diethylene glycol a significant higher extent of degradation was found in the Oxitop. The difference of the mean values for NTA was >10% but due to the high standard deviation of the data the mean values did not prove to be significantly different (Students t-test, 95% confidence level). The lag phases in the two different systems were quite consistent, with NTA being the only exception. The k1 constants varied in a certain range but no system showed constantly higher or lower values than the other one. Typically, k1 constants lay in the range of 0.047–0.934 day1, with most data being in a range from 0.10 to 0.30 day1 (Table 3). 3.4. Degradation of aniline in different biodegradation tests
The degradation pattern of ten different chemical compounds was studied in both respirometer systems. Parameters determined were the lag phase, the k1 constants for determining the degradation rate, and the extent of biodegradation, usually expressed as a percentage. The data obtained are summarised in Table 2. In eight cases the extent of degradation was in the same range (difference p10%); in one case (2-ethylhex-
Since aniline is a widely used reference substance for biodegradation tests, its degradation in different OECD tests has been extensively evaluated over many years. The test systems used were the Zahn-Wellens test for comparative purposes (OECD 302 B, ISO 9888), which is a test for inherent biodegradability, the DOC dieaway test (OECD 301 A, ISO 7827), the CO2 evolution
Table 1 BOD data from blank values derived from the Oxitop and Sapromat system. The number of data analyzed was 30 for the Oxitop system and 146 for the Sapromat Time
Test system Oxitop
Day 6 Day 14 Day 28
Sapromat
Mean
St. Dev.
SE Mean
Mean
St. Dev.
SE Mean
11.13 18.60 26.00
4.84 5.77 7.62
1.25 1.49 1.97
10.06 15.34 20.00
3.55 4.69 6.59
0.29 0.40 0.55
St. Dev., standard deviation; SE Mean, standard error of the mean.
Table 2 Influence of different inocula on the degradation of cyclohexanone and ethylhexylacrylate Test compound
Parameter
Oxitop
Sapromat
Municipal sludge
Industrial sludge
Municipal sludge
Industrial sludge
Cyclohexanone
Extent of degradation (%) Lag period (days) k1 -constant (day1)
91 (1.6) 0.5 (0.1) 0.224 (0.006)
91 (1.0) 0.5 (0.1) 0.126 (0.005)
96 (6.7) 0.5 (0.1) 0.216 (0.065)
83 (12.2) 0.5 (0.1) 0.162 (0.019)
2-Ethylhexylacrylate
Extent of degradation (%) Lag period (days) k1 -constant (day1)
58 (7.6) 1 (0.1) 0.047 (0.008)
56 (2.1) 1 (0.2) 0.058 (0.003)
73 (7.5) 1 (0.2) 0.122 (0.012)
71 (3.1) 1 (0.2) 0.106 (0.013)
The standard deviation of the results is given in brackets. Tests were performed in triplicate.
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3.5. Nitrification processes and their inhibition
test (OECD 301 B, ISO 9439) and the manometric respirometry test (OECD 301 F, ISO 9408). Again the Sapromat and Oxitop systems were both used in parallel. The data summarised in Table 4 show that the k1 constants are comparable in both respirometric systems, whereas the k1 constants in the DOC die-away test are the highest and in the CO2 evolution test, the lowest. The lag periods are comparable in the Oxitop system, the DOC die-away test and the CO2 evolution test, but longer in the Sapromat test system. Also, the final extent of degradation is comparable in the Oxitop, the DOC die-away test and the CO2 evolution test, whereas in the Sapromat a lower extent of degradation is observed. When testing aniline degradation in the Oxitop with different carbon dioxide absorbing materials (NaOH, KOH and sodium lime) it was found that similar degradation curves were obtained.
Nitrification processes have a striking effect on the biochemical oxygen demand, because 1 g of organic nitrogen released as ammonium is equivalent to 4.57 g of oxygen. In general, the ammonium sources in biodegradation tests are the mineral medium and the inoculum, but depending on the test conditions and the test duration also nitrogenous organic compounds can contribute to the nitrification and hence influence the test results of respirometric tests. This can be compensated for by either subtracting the oxygen in the resultant nitrate and nitrite from the measured BOD values as it is foreseen in the standard method or by preventing nitrification by the addition of a specific inhibitor such as allylthiourea (ATU). As can be seen in Figs. 2 and 3 a concentration of 10 mg l1 ATU
Table 3 Degradation of different chemical compounds in respirometric test systems Test compound
Test system Sapromat
Acrylic acid Benzoic acid Diethylene glycol Glycerol Morpholine NTA 1.5-Pentanediol Phenol
Oxitop
Degradation (%)
k1 -constant (day1)
Lag period (days)
Degradation (%)
k1 -constant (day1)
Lag period (days)
94 91 59 68 87 81 83 81
0.934 0.556 0.081 0.258 0.120 0.214 0.342 0.224
2.5 (0.3) 0 (0.1) 5 (1.0) 1 (0.25) 16 (2.2) 14 (2.08) 3 (0.45) 3 (0.45)
91 93 89 78 89 70 89 76
0.993 0.461 0.173 0.200 0.248 0.564 0.222 0.412
3 (0.2) 0 (0.1) 5 (1.0) 1 (1.0) 16 (1.8) 9 (1.0) 3 (0.3) 3 (0.3)
(3.78) (9.09) (12.03) (5.29) (5.77) (11.06) (8.51) (2.52)
(0.29) (0.17) (0.026) (0.083) (0.038) (0.064) (0.11) (0.06)
(12.06) (0.83) (3.63) (2.89) (5.77) (10.23) (6.11) (12.19)
(0.32) (0.14) (0.04) (0.04) (0.06) (0.12) (0.05) (0.10)
The number of replicate tests performed was 3 (acrylic acid, glycerol, morpholine, 1.5-pentanediol, phenol) to 6 (benzoic acid, diethylene glycol, NTA). The standard deviation of the results is given in brackets.
Table 4 Degradation of aniline in different OECD biodegradation tests and in the CO2 headspace test Test system
Parameter k1 -constant (day1)
Lag period (days)
Extent of degradation (%)
Number of tests performed
OECD 301 DOC-die-away test OECD 301 B Sturm test OECD 301 F Oxitop OECD 301 F Sapromat CO2-headspace test
1.71 0.30 0.74 0.86 n.d.
2.2 (0.94) 3.0 (0.71) 3.6 (0.8) 6.0 (2.1) n.d.
91 51 13 150 22
OECD 302 B Zahn-Wellens test
0.096
96 87 92 81 87 97 98
a
(0.55) (0.12) (0.25) (0.21)
CO2 evolution. DOC elimination. n.d. not determined. The standard deviation of the results is given in brackets. b
3
(3.0) (9.0) (12.8) (11.4) (6.0)a (3.0)b
1
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1.6
1.6
without ATU with ATU
1.2 1 0.8 0.6 0.4 0.2
(A)
without ATU with ATU
1.4 Degradation (BOD/ThOD)
Degradation (BOD/ThOD)
1.4
0
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1.2 1 0.8 0.6 0.4 0.2
0
5
10
15 20 Time (days)
25
0
30 (B)
0
5
10
15 20 Time (days)
25
30
Fig. 2. Influence of allylthiourea (ATU) on the oxygen demand during the degradation of aniline (A) and isopropylamine (B) in the Oxitop system.
Fig. 3. Influence of allylthiourea (ATU) on the oxygen demand during the degradation of aniline (A) and isopropylamine (B) in the Sapromat system.
effectively inhibits nitrification. At this concentration the heterotrophic respiration is not effected. Nitrification processes occur in most OECD 301 tests, the extent differs, however. Aniline, for example, was usually not nitrified in the Sapromat whereas it was nitrified in the Oxitop without addition of ATU. The measured concentrations of nitrite and nitrate ranged from about 0.02 to 0.9 mM after an incubation period of 28 days (Fig. 4). The reason for the missing nitrification still remains unclear. For all other test conditions were identical, it can only be due to the different construction of the respirometers. The isopropylamin nitrogen, on the other hand, was nitrified in both respirometer systems.
3.6. Biodegradation modelling Different mathematical approaches were used to model the biodegradation kinetics data obtained. The models used were a modified logistic plot and a firstorder plot. An example for the biodegradation of morpholine and diethylene glycol is shown in Figs. 5A–D and 6A–D. It is obvious that the modified logistic plot can be used for all sigmoidal biodegradation curves, whereas the first-order plot is suitable for biodegradation curves with flat lag periods. On the other hand, the first-order plot is a tool that can be used to determine easily the half-time constant of a chemical compound. The prerequisite for the use of a first-order
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Fig. 4. Nitrification in different biodegradation test systems. The formation of nitrate/nitrite-N is illustrated in blank controls (A) and degradation assays containing aniline (B). The mean values of 7 to 14 investigations are shown. The test systems used are (1) DOC dieaway test (OECD 301 A), (2) CO2 evolution test (OECD 301 B), (3) manomeric respirometry test (Sapromat, OECD 301 F), (4) manomeric respirometry test (Oxitop, OECD 301 F), (5) CO2-headspace test.
plot is that there are enough data in the exponential phase (in general to 6 8 data points) to yield sufficient accuracy.
4. Discussion According to OECD guidelines and ISO standards blank values for endogenous respiration should be in the range of 20–30 mg l1 and not exceed 60 mg l1. Otherwise the experimental technique has to be examined or the test repeated with another inoculum [3] The data reported in this study are in the given range for both respirometric systems. Endogenous respiration processes are due to aerobic biodegradation of residual carbon sources of the inoculum and self-digestion processes present. A range of 20–30 mg l1 BOD in the blank is equivalent to a ThOD of 7.5 to 11.25 mg l1 of carbon in glucose (0.6–0.9 mM of carbon). When comparing the ThOD or the chemical oxygen demand (COD) added to the test system at the recommended test concentrations (100 mg l1 substance or 50–100 mg l1 ThOD), the endogenous respiration represents a fraction of 0.2–0.6 of the total oxygen demand and is therefore significant. The most convenient way to reduce endogenous respiration is to starve the inoculum. The drawback of this method is the possible reduction in the number of viable bacterial cells and subsequently the loss of biodegradation potential. A comparison of the two different respirometric test systems with numerous test substances revealed that both systems can give different data. The advantage of the Oxitop is its compact design, the accuracy and
reliability of the measuring units, the easy reading of the measured data and its transfer to a computer and a relatively low price. The main advantage of the Sapromat is the oxygen replacement and the option possibility to extend the duration nearly infinitely. Another important point in biodegradation tests is the source of the inoculum. It is known that inocula from industrial wastewater treatment plants show generally a higher biodegradation potential for chemical compounds than inocula from municipal wastewater treatment plants. In this study, we were, however, unable to confirm this assumption. For example, the degradation rate of cyclohexanone was faster with municipal activated sludge than with industrial activated sludge. On the other hand, with 2-ethylhexylacrylate, inocula from both sources showed comparable biodegradation rates. In the case of morpholine, lag phases of 15–16 days were observed before the onset of biodegradation processes. This extended adaptation period has also been reported in former studies [13]. Therefore, it is possible that, due to the extended lag phase and the low inoculum concentrations in OECD 301 tests, the number of bacteria may not always be sufficient for immediate biodegradation. However, a mixture of different inocula may deliver the sufficient diversity and the use of a degradable substrate in addition to the test substance may enable cometabolism to eventually bring about complete biodegradation of recalcitrant test substances. These facts underline the stringency of the OECD 301 tests. This stringency of the test conditions together with alterations in the metabolic activity of the inoculum used may also be the reason for the variations in biodegradability as observed in both respirometric test systems. By
P. Reuschenbach et al. / Water Research 37 (2003) 1571–1582
Oxitop (k1=0.248 day-1)
Oxitop (k = 0.76 day-1) Sapromat (k = 0.46 day-1)
Sapromat (k1=0.120 day-1) 1
1
0.8
0.8 Degradation
Degradation
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0.6 0.4
0.6 0.4 0.2
0.2
0
0 15
20 25 Time (days)
(A)
0
30
5
10
15
20
25
30
Time (days)
(B)
Oxitop (k = 0.43 day-1) Sapromat (k = 0.15 day-1)
Oxitop (k1=0.173 day-1) Sapromat (k1=0.081 day-1)
1
0.9
0.8 Degradation
0.8
Degradation
0.7 0.6 0.5 0.4
0.6 0.4
0.3
0.2
0.2
0
0.1 5
(C)
10
15
20
25
0
30
Time (days)
(D)
5
10
15
20
25
30
Time (days)
Fig. 5. Kinetical analysis of the degradation of morpholine (A,B) and diethylene glycol (C,D). The data were analysed with first-order plots (A, C) and a modified logistic function (B,D).
parallel testing with a recommended control substance (aniline) major alterations in the quality of the inoculum used are detected. The suboptimal test conditions of the OECD 301 tests especially for adaption processes have also been recognised by the OECD, but are regarded as necessary for a realistic simulation of biodegradation in surface water. In this way variations of the test results may reflect variations in biodegradation pattern under realistic environmental conditions. In our investigations we found that the k1 constants for aniline were highest in the DOC die-away test and lowest in the CO2 evolution test (Table 4). This observation is of general interest for the determination of ready biodegradability according to the OECD criteria and may be explained as follows. The DOC removal from water by adsorption on the biomass or by uptake into the cells is the first step and has therefore the
highest rate. The consumption of oxygen for oxidative processes and finally the production of carbon dioxide are subsequent steps with lower rates. DOC removal and the depletion of oxygen in the medium can be monitored directly in the test vessel, whereas the carbon dioxide is measured in the traps outside the test medium. Due to the necessary liberation of carbon dioxide from the liquid phase in the test vessel and the subsequent diffusion processes into the trap, lower biodegradation is reasonable for this type of test. A disadvantage of test systems based on BOD and CO2 is the difficulty to differentiate between the oxidised part of the test substance and the part incorporated in biomass which can be expressed by the heterotrophic yield coefficient [14,15]. Heterotrophic yield coefficients can vary between 0.40 and 0.80 and are dependent from the substrate used, the source of the inoculum, the ratio
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municipal inoculum (k = 0.63 day-1) industrial inoculum (k = 0.24 day-1)
municipal inoculum (k = 0.32 day-1) industrial inoculum (k = 0.18 day-1)
1
1
0.8 Degradation
Degradation
0.8 0.6 0.4
0.6
0.4
0.2
0.2 0
0
5
(A)
10
15
20
25
0
30
0
15
20
25
30
municipal inoculum (k1 = 0.190 day-1)
industrial inoculum (k1 = 0.129 day-1)
industrial inoculum (k1 = 0.145 day-1)
1
1
0.8
0.8 Degradation
Degradation
10
Time (days)
municipal inoculum (k1 = 0.221 day-1)
0.6 0.4
0.6 0.4 0.2
0.2
0
0 0
(C)
5
(B)
Time (days)
5
10
15
20
25
30
Time (days)
0
(D)
5
10
15
20
25
30
Time (days)
Fig. 6. Degradation of cyclohexanone in the Oxitop (A, C) and Sapromat system (B, D). Municipal and industrial inocula were used for these biodegradation studies. The data were analysed with first-order plots (C,D) and a modified logistic function (A, B).
of substrate to inoculum and the test conditions used. Therefore, higher extents of degradation in tests based on carbon removal in comparison to tests based on oxygen consumption and carbon dioxide formation can be explained in this way. In general, extents of biodegradation of >60% (BOD of THOD or CO2 of THCO2) indicate a sufficient biodegradation [3]. Test results based on DOC removal, on the other hand, seem to be much more reliable as degrees of >90% indicate good degradation. This discrepancy could be solved either by performing expensive carbon balances, which allow a clear understanding of the fate of the test substance or by the use of limit values and degradation criteria. Experts have defined the OECD criteria for ready biodegradability and if they are fulfilled the test substance is from definition biodegradable. The biodegradation patterns or curves of standardised batch tests are described by the lag phase, the final extent of biodegradation in the plateau phase and the time taken to reach the plateau. A modern approach to describing
biodegradation kinetics is the use of mathematical models. A variety of such methods exist which can be applied [1]. In this study we focussed on two principal procedures. In first-order reactions the rate of biodegradation is only proportional to the concentration of the test substance. Linear regression procedures are usually used to determine the first-order rate constant k1 ; but it is also possible to use non-linear procedures [10,11,16,17]. In the literature, a number of k1 constants have been published for different industrial chemicals such as benzene [18], chlorobenzene [19], 4-chlorophenol [18], methyl parathion [20], nitrilotriacetic acid (NTA) [17,21,22]. The first-order rate constants range from 0.01 to 4.8 day1. Most of the differences reported were due to the quantity and quality of the biomass used [1]. To calculate first-order kinetics the determination of sufficient biodegradation data in the degradation phase is necessary. In order to model the biodegradation curve from the lag phase to the plateau phase, the modified logistic model was used. This mathematical model is
P. Reuschenbach et al. / Water Research 37 (2003) 1571–1582
based on a logistic function, which is often used to describe the population pattern under growth limiting conditions [23]. Other methods frequently used are the Monod growth model with and without growth of biomass, the zero order model and the logarithmic model. A critical discussion of these models was published by Simkins and Alexander [24]. For future standards it could be advantageous to integrate these modern mathematical tools into standardised methods. For example, the first-order kinetic model could be used instead of the 10-day-window to describe degradation kinetics, and the modified logistic model could be appropriate in estimating the lag phase as well as the final extent of degradation. There are already ISO standards available which are used to determine biodegradation kinetics (ISO 14592 parts 1 and 2) indicating the positive future perspectives of biodegradation kinetics.
5. Conclusions The Oxitop and Sapromat method were found to be reliable respirometric test methods for assessing the biodegradability of chemical compounds. In the Oxitop system endogenous BOD production proved to be significantly higher after 14 and 28 days of incubation. On the other hand after 6 days of incubation the differences were not significant. When testing municipal and industrial activated sludge as inocula minor differences in the extent of biodegradation could be stated for cyclohexanone and 2-ethylhexylacrylate. On the other hand, the degradation rate of cyclohexanone was higher with a municipal inoulum. Both respirometric systems were used to evaluate the biodegradability of different chemical compounds. In eight cases comparable results were found. In two cases (diethylene glycol and 2-ethylhexylacrylate) different results were obtained indicating that the test systems are not always equivalent. Aniline is a recommended reference compound for biodegradation tests. In different OECD and ISO test systems it was found that in the Sapromat longer lag periods and a lower extent of biodegradation occurred. As expected the degradation rate constant k1 was highest in the DOC die-away test (OECD 301A) and lowest in the CO2 evolution test (OECD 301B). Allylthiourea (ATU) at a final concentration of 10 mg l1 can effectively inhibit nitrification processes which may occur during biodegradation studies. At this concentration the heterotrophic degradation activity is not disturbed. In nearly all OECD and ISO test systems nitrification processes were observed. Only when testing aniline in the Sapromat system no nitrification could be found.
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Biodegradation modelling could be performed by using a first-order plot for biodegradation curves with a flat lag period and a modified logistic plot for sigmoidal biodegradation curves.
References [1] Battersby NS. A review of biodegradation kinetics in the aquatic environment. Chemosphere 1990;21:1243–84. [2] Swisher RD. Surfactant biodegradation, 2nd ed. New York: Marcel Dekker Inc., 1987. [3] OECD Guidelines. OECD Guidelines for testing of chemicals, Paris, 1993. OECD 301 A: DOC Die-AwayTest; OECD 301 B: CO2 Evolution Test; OECD 301 C: Modified MITI Test (I); OECD 301 D: Closed Bottle Test; OECD 301 E: Modified OECD Screening Test; OECD 301 F: Manometric Respirometry Test; OECD 302 B: ZahnWellens/EMPA test. [4] ISO (International Organization for Standardization). ISO 7827 (1994): Water Quality—Evaluation in an aqueous medium of the ‘‘ultimate’’ aerobic biodegradability of organic compounds—method by analysis of dissolved organic carbon; ISO 9439 (1999): Water Quality—Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium—Carbon dioxide evolution test; ISO 9408 (1999): Water Quality—Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium by determination of oxygen demand in a closed respirometer; ISO 9888 (1999): Water Quality— Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium—Static test (Zahn-Wellens method); ISO 10707 (1994): Water Quality—Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium—method by analysis of biochemical oxygen demand (closed bottle test); ISO 10708 (1997): Water Quality—Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium—method by determining the biochemical oxygen demand in a two-phase closed bottle test; ISO 14592 (draft): Water Quality—Evaluation of the aerobic biodegradability of organic compounds at low concentrations— Part 1: Shake flask batch test with surface water or surface water sediment suspensions—Part 2: Continuous river flow model with attached biomass; ISO 14593 (1999): Water Quality—Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium—Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test); ISO 15462 (1997): Water Quality— Selection of tests for biodegradability (technical report). [5] Pagga U. Testing biodegradability with standardized methods. Chemosphere 1997;35:2953–72. [6] Conzelmann F, Beitlich R. Einfache Bestimmung der biologischen Abbaubarkeit nach OECD 301 F mit dem Oxitop (A simple determination method of biodegradability according to OECD 301 F with the Oxitop system). Melliand, October 1996. p. 700. [7] Govind R, Gao C, Lai L, Tabak HH. Continuous, automated and simultaneous measurement of oxygen uptake and carbon dioxide evolution in biological systems. Water Envrion Res 1997;69(1):73.
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[17] Larson RJ. Kinetic and ecological approaches for predicting biodegradation rates of organic chemicals in natural ecosystems. In: Klug MJ, Reddy CA, editors. Current perspectives in microbial ecology. Washington: American Society for Microbiology, 1984. p. 677–86. [18] Lee RF, Ryan C. Microbial degradation of organochlorine compounds in estuarine waters and sediments. In: Bourquin AW, Pritchard PH, editors. Proceedings of the Workshop Microbial Degradation of Pollutants in Marine Environments: EPA 600/9-79-012, 1979. p. 443–50. [19] Bartholomev GW, Pfaender FK. Influence of spatial and temporal variations on organic pollutant biodegradation rates in an estuarine environment. Appl Environ Microbiol 1983;45:103–9. [20] Cripe CR, Walker WW, Pritchard PH, Bourqui AW. A shake-flask test for estimation of bodegrdability of toxic organice substances in the aquatic environment. Ecotoxicol Environ Saf 1987;14:239–51. [21] Larson RJ. Comparison of biodegradation rates in laboratory screening studies with rates in natural waters. Residue Rev 1983;85:159–71. [22] Larson RJ, Ventullo RM. Kinetics of biodegradation of nitrilotriacetic acid (NTA) in estuarine environment. Ecotoxicol Environ Saf 1986;12:166–79. [23] Finney RL, Weir MD, Giordano FR. Thomas’ calculus, 10th ed. Boston, San Francisco, New York: Addison Wesley, Longman, 2001. p. 514–518. [24] Simkins S, Alexander M. Models for mineralisation kinetics with variables of substrate concentration and population density. Appl Environ Microbiol 1984;47: 1299–306.