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Determination of the aerobic biodegradability of polymeric material in aquatic batch tests
Chemosphere 42 (2001) 319±331
Determination of the aerobic biodegradability of polymeric material in aquatic batch tests Udo Pagga
a,*
, Anja Sch afer b, Rolf-Joachim M uller c, Michael Pantke
d
a
BASF Aktiengesellschaft Ecology, D-67056 Ludwigshafen, Germany Endress und Hauser, Dieselstr. 24, D-10839 Gerlingen, Germany (formerly University Stuttgart) Gesellschaft f ur Biotechnologische Forschung mbH Mascheroder Weg 1, D-38124 Braunschweig, Germany d Bundesanstalt f ur Materialpr ufung Unter den Eichen 87, D-12205 Berlin, Germany b
c
Received 27 September 1999; accepted 8 February 2000
Abstract Results of an international ring-test of two laboratory methods are presented for investigating the biodegradability of organic polymeric test materials in aquatic test systems based on respirometry and the evolution of carbon dioxide. These methods are developed further from the well-known standardized biodegradation tests ISO 9408 (1999) and ISO 9439 (1999), which have been successfully used for many years. The most important improvements are the extension of the test period up to six months, the increase of the buer capacity and nutrient supply of the inorganic medium, an optimization of the inoculation, and optionally, the possibility of a carbon balance. A ring test, organized by the International Biodeterioration Research Group (IBRG), was run using a poly(,-caprolactone)-starch blend and an aliphatic-aromatic co-polyester as test materials and a microcrystalline cellulose powder as a reference material. The test results and the experience gained by the participants showed that the methods are suitable and practicable. The test methods have been meanwhile established as standards ISO 14851 (1999) and ISO 14852 (1999). Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Aerobic biodegradability; Respirometry; Carbon dioxide evolution; Polymeric materials; Cellulose powder
1. Introduction Composting of solid waste has been recognised as a valuable method for waste treatment. Typical input materials are not only green waste from gardens, biowaste from households and natural materials such as paper, but also manufactured products, for example waste bags, which are more and more based on biodegradable plastics to allow this biological waste
*
Corresponding author. Tel.: 621-60-58148; fax: 621-6058043. E-mail address: [email protected] (U. Pagga).
treatment. Compostable products, especially synthetic plastic materials, need to be identi®ed and classi®ed as being biodegradable and must not adversely in¯uence the composting process or the quality of the compost produced (Pagga et al., 1996; Pagga, 1997a, 1998). Test methods and test strategies for this purpose have been developed and recently standardized on national (e.g., in Germany DIN 54900, 1998) and European level (EN 13432 (Draft 1999)) and worldwide (ISO, 15986). As biodegradability is a basic prerequisite for chemicals in general a number of biodegradation tests have been developed and standardized in the last 25 years (ISO 15462, 1997; Pagga, 1997b). Most of them are intended to determine aerobic biodegradability of
0045-6535/01/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 0 ) 0 0 0 6 9 - 2
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organic chemicals in aquatic batch systems such as the well-known and much-used manometric respirometry test (OECD 301 F, 1993; ISO 9408, 1999), the twophase closed bottle test (ISO 10708) or the CO2 evolution test (OECD 301 B, 1993; ISO 9439, 1999). But only recently have special tests for polymeric materials been suggested and standardized. One is the aerobic composting test (Pagga et al., 1995; ISO 14855, 1999); further possibilities are specialised aquatic screening tests. The usual aquatic biodegradation tests have a considerably lower degradation potential than terrestrial tests, because fungi and actinomycetes, which are very important for polymer degradation, do not have optimum growth conditions in water. The standard test duration of 28 days applied in such tests is also too short for polymeric test materials. Therefore the respirometric test (P uchner et al., 1995) and the CO2 evolution test (Urstadt et al., 1995a; Sch afer, 1999) have been modi®ed and improved for this speci®c application by using, for example, suspensions of compost as inoculum, inorganic media with higher buering capacity and more nutrients, and by the extending of the test duration and the temperature range. The advantage of the aquatic tests is that they have a somewhat lower requirement of resources for the study and they can be used to prepare a carbon balance which characterises the extent of biodegradation better than the CO2 evolution alone, particularly if a signi®cant amount of biomass is formed during the test. A carbon balance is not possible in a terrestrial test using compost because there is too much organic carbon in the compost. The use of the synthetic organic carbon-free mineral medium in the aquatic test avoids the so-called priming eect, a stimulation of the degradation by addition of plastic samples to a compost which cannot be corrected by the blank values. These aquatic tests provide, however, in the case of plastic material in ®rst line basic information on biodegradation and do not have the character of a simulation test for an aquatic environment. The biodegradation in a solid compost matrix at temperatures up to 60°C may be better simulated in the aerobic composting test (Pagga et al., 1995; ISO 14855, 1999). The Plastic Committee TC61 of the International Standardization Organization (ISO) decided to standardize these aquatic tests and working group 22 of ISO TC 61/SC 5 had the task of preparing the International Standards. In the course of standardization a ring test was performed with the principal aim of investigating the suitability of the methods for testing plastic materials and of allowing a number of laboratories to obtain more practical experience. The test was organized by the Biodegradable Plastics Group of the International Biodeterioration Research Group (IBRG). This publication describes the test methods and presents the results of the participants of the ring test.
2. Test methods The International Standards ISO 14851 (1999) and ISO 14852 (1999) are specially designed for determining the aerobic biodegradability of polymeric and plastic materials including formulation additives. In both tests the test material is exposed under laboratory conditions in an aqueous medium to an inoculum with a wide variety of microorganisms which is either activated sludge or a compost suspension. According to the test procedure activated sludge is collected from a well-operated sewage treatment plant or a laboratory plant and used in a concentration range of 30±1000 mg/l suspended solids in the ®nal test mixture. When biodegradation processes in a natural aquatic environment should be simulated or a carbon balance is carried out, an inoculum concentration of 30 mg/l is recommended. In the case of a compost inoculum 10 g of non-sterile compost from a composting plant treating predominantly organic waste are suspended in 100 ml of inorganic test medium. The supernatant is used after settling or ®ltration at a recommended concentration of 1±5% (v/v) in the test ¯asks. The use of compost can increase the number of fungi in the test ¯asks and improve the biodegradation of plastic materials. Instead of compost fertile soil may be used as well. The inorganic salts of the medium can optionally be used in a concentration about ten-fold higher than the usual standardized biodegradation tests (see ISO 15462, 1997). This is to improve the buer capacity of the medium and the available amount of inorganic nutrients especially nitrogen and phosphorus salts. The test duration is extended to up to six months compared to usually four weeks in standard aquatic biodegradation tests. The temperature may be chosen in a broad range to allow especially the growth of thermophilic microorganisms which may have a better degradative potential for polymers than mesophilics. ISO 14851 (1999) speci®es a method by determining the biochemical oxygen demand (BOD) in a closed respirometer. The test mixture of test material, inorganic medium and inoculum is stirred in closed ¯asks of the respirometer (Fig. 1). Evolved carbon dioxide (CO2 ) is absorbed in a suitable absorber (e.g., soda lime) in the head space of the test ¯asks. The oxygen consumption is determined by measuring the amount of oxygen required to maintain a constant gas volume in the respirometer ¯ask, or by measuring the change in volume or pressure either automatically or manually. The level of biodegradation is determined by comparing the BOD with the theoretical oxygen demand (ThOD) and expressed in percent. The in¯uence of possible nitri®cation processes on the BOD has to be considered in cases of polymers containing nitrogen. A variation of this test which needs no expensive respirometer is described in annex D of ISO 14851
U. Pagga et al. / Chemosphere 42 (2001) 319±331
321
Fig. 1. Principle of a closed respirometer ± ISO 14851 (1999) (example).
(1999). The method is identical to the two-phase closed bottle test ISO 10708. The inoculated test mixture is shaken or stirred in the test bottles. Known volumes of medium and air are used to assure steady-state oxygen partitioning between liquid and gas phases. The degradation is followed by regularly measuring the dissolved oxygen concentration in the aqueous phase usually by an oxygen electrode. The oxygen uptake is calculated from the measured dissolved oxygen concentrations divided by the oxygen saturation value under normal conditions and multiplied by the total oxygen content originally present in the liquid and gaseous phases. Biodegradability is then calculated and expressed as in the case of the respirometer. The second aquatic test, ISO 14852 (1999) is based on the determination of the evolved CO2 (Fig. 2). The same test mixture as for ISO 14851 (1999) is agitated in test ¯asks and aerated with CO2 -free air over the test period.
The CO2 evolved during the microbial degradation which is in the exhaust air is determined by suitable methods. Such methods are, for example, absorption of CO2 in sodium hydroxide solution and determination of dissolved inorganic carbon (DIC) in a carbon analyzer, absorption of CO2 in a barium hydroxide solution and subsequent titration, gas chromatography or continuous infrared detection. The level of biodegradation is calculated by comparing the evolved CO2 with the theoretical amount (ThCO2 ) and again expressed in percent. The process of biodegradation is shown in both tests in a curve where either BOD or CO2 production or the calculated biodegradation percentage is plotted as a function of time. Typical biodegradation curves consist of a lag phase, which is the time from the start of a test until a distinct biodegradation (e.g., 10%) can be measured, a biodegradation phase, in which the maximum degradation takes place and a plateau phase, in which it
Fig. 2. Principle of the CO2 evolution test ± ISO 14852 (1999) (example).
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is almost completed. The test result is the biodegradation degree in percent, calculated as a mean value from the measured data of the plateau phases of parallel test vessels. The methods described here are used to determine the ultimate biodegradation which is the complete utilisation of a test material by microorganisms resulting in the production of carbon dioxide, water, mineral salts and new microbial cellular constituents (biomass) in contrast to a primary biodegradation which is only the change of the identity of the test material (Pagga, 1999). As an option a carbon balance may be performed and calculated to obtain additional information on biodegradation. Plastic materials are normally of more complex composition than substances with low molecular weights. Therefore determination of CO2 evolution or of BOD alone may not be sucient to characterize and quantify their biodegradability. During biodegradation new biomass is built up by the microorganisms and some of the organic carbon of the test material used is transformed to biomass but not biochemically oxidised. That is the reason why the analytical parameter CO2 evolution and BOD will often not reach 100% of the theoretical values ThCO2 and ThOD even in the case of complete biodegradation of a test material and insucient degradation could falsely be assessed from the test results. A balance of the fate of the organic carbon of the test material may be helpful to con®rm complete biodegradability. The principle of such a balance is the detailed quantitative measurement of the CO2 evolved (either directly determined in the exhaust air or calculated from the measured BOD), the new produced biomass (determined, e.g., as protein), the water soluble metabolites generated during the biodegradation process (determined as dissolved organic carbon DOC), and the residual undegraded polymeric test material using preferably a substance speci®c analytical method. The measured values are transformed to carbon and the sum is compared with the amount of the organic carbon of the test material introduced into the test system. As an option a carbon balance may be performed and calculated to obtain additional information on biodegradation. Plastic materials are normally of more complex composition than substances with low molecular weights. Therefore the determination of CO2 evolution or of BOD alone may not be sucient to characterize and quantify their biodegradability. During biodegradation new biomass is built up by the microorganisms and some of the organic carbon of the test material used is transformed to biomass but not biochemically oxidised. Therefore analytical parameters such as CO2 evolution and BOD will often not reach 100% of the respective theoretical values even in the case of complete biodegradation of a test material and an insucient degradation could falsely be evaluated from the test results. A balance of the fate of the organic carbon of the test material may be helpful to con®rm
complete biodegradability. The principle of such a balance is the detailed quantitative measurement of the CO2 evolved (either directly determined in the exhaust air or calculated from the measured BOD), the new produced biomass, the water soluble metabolites generated during the biodegradation process (determined as dissolved organic carbon DOC), and the residual undegraded polymeric test material. The measured values are transformed to carbon and the sum is compared with the amount of the organic carbon of the test material introduced at the beginning into the test system. Two steps of the carbon balance are crucial, the determination of the biomass and the residual polymers. Biomass may be determined at the beginning and the end of the test in the inoculum using suitable methods, e.g., by protein measurement. From the measured value the amount of carbon in the biomass is determined. From the dierence the increase of organic carbon of the biomass is calculated. The residual polymers can be extracted at the end of the test in the total remaining samples and directly determined if any polymer speci®c analysis is available. From the usually known composition of the polymers the amount of carbon can be calculated. If no suitable analytical technique is available also an indirect determination is possible, e.g., by washing, drying and weighing the residue and determining the total organic carbon (TOC). From this TOC the organic carbon of the biomass is subtracted to obtain the amount of carbon of the residual polymers. 3. Test materials Mater Bi ZI01U a poly(,-caprolactone)-starch blend obtained from Novamont S.P.A., Novara, Italy, with a mean particle size of <180 lm and the following elemental composition: carbon 53.6%; hydrogen 8.0%; nitrogen 0%; oxygen 38.4% (w/w) as stated by the supplier. ABA 618 an aliphatic-aromatic copolyester with the following elemental composition: carbon 61.7%; hydrogen 8.8%; nitrogen 5.7%; oxygen 23.8% (w/w) and with a mean molar mass of a monomer of 147.8 as stated by the supplier. As a positive control for the check of the activity of the inoculum during the test a microcrystalline cellulose powder, Avicel, was used with the following elemental composition: carbon 41.8%; hydrogen 6.4%; nitrogen 0%; oxygen 52.0% (w/w) as measured by BASF. Avicel (Nr. 2330) was obtained from Merck, Darmstadt, Germany. 4. Results The results of the ring test are shown in Tables 1±7 and Figs. 3±8. The carbon balance is just based on BOD
U. Pagga et al. / Chemosphere 42 (2001) 319±331
323
Table 1 ISO 14851 (1999) ± Avicel (reference substance) Laboratory
Degree of degradation (% ThOD)
Degradation based on Cbalance (%)
Input material (mg/l)
Test duration (d)
Temperature (°C)
pHend with test material
pHend blank
Inoculum
1 2
69 79
± ±
± 100
28 28
25 25
6.8 7.4
7.6 7.5
3
84
100
500
40
20
7: 0
7: 0
4
82 67
90 86
160
41
35
7.0 9.6
7.0 8.6
Compost suspension Activated sludge (30 mg/l MLSS) Activated sludge 1% (v/v)
5
79
100
62
20
±
±
6
61 61 73
67
100
47
20
7.2
7.2
70
65
7.2
7.2
±
Compost suspension 2% (v/v) Compost suspension 1.5% (v/v) Compost suspension 3% (v/v)
Table 2 ISO 14851 (1999) ± Mater-Bi ZI01U
a
Laboratory
Degree of degradation (% ThOD)
Degradation based on Cbalance (%)
Input material (mg/l)
Test duration (d)
Temperature (°C)
pHend with test material
pHend blank
Inoculum
1 3
63a 76
±
82
1000 400
28 36
25 20
7.1 7.0
7.6 7.0
4
80
93
160
41
35
8.7
8.6
5
50
100
62
20
±
±
6
44 59 60
Compost suspension Activated sludge 1% (v/v) Compost suspension 2% (v/v) Compost suspension 1.5% (v/v)
102
100
47
20
7: 2
7: 2
47 60
83 103
7.2 7.2
7.2
±
Compost suspension 3% (v/v)
Plateau phase not reached.
or CO2 evolution and the determination of biomass. The source and concentration of the inoculum is indicated as given by the participating laboratories (MLSS mixed liquor suspended solids). 4.1. ISO 14851 respirometric test The degrees of biodegradation of the reference substance Avicel were 61±84% based on ThOD and 65± 100% including carbon balance (Table 1, Fig. 3). The course of the degradation curves are comparable, particularly taking into account the dierent test temperatures and the dierent inocula used. The lag phases in all tests were coherently around ®ve days. Dierences increased with progressing degradation, however, all
degrees of degradation at the end of the tests exceeded the limit value of 60% which is requested by the standard to prove the validity of the test. Some tests were ®nished before a clear plateau was reached, in these cases higher ®nal degrees of degradation could be expected. With the exception of one laboratory, the carbon found in the biomass as one part of the carbon balance contributed signi®cantly to the degree of biodegradation. The degrees of biodegradation of Mater Bi ZI01U were 44±80% based on ThOD and 82±103% including carbon balance (Table 2, Fig. 4). The variation between the degradation curves is greater than in the case of Avicel. The observed degrees of degradation are comparable with those found with the positive control, but
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Table 3 ISO 14851 (1999) ± ABA 618
a
Laboratory
Degree of degradation (% ThOD)
Degradation based on Cbalance (%)
Input material (mg/l)
Test duration (d)
Temperature (°C)
pHend with test material
pHend blank
Inoculum
1 2
4 77a
±
98
1000 100
28 28
25 25
7.7 7.2
7.6 7.2
3
7 14
104 22
500
40
20
7.4 7: 0
7.4 7: 3
Compost suspension Activated sludge (30 mg/l MLSS)
4
31
40
160
41
35
8: 6
8: 7
5
23
100
62
20
±
±
6
11 7
11
100
47
20
7.1
7.2
3 17
4 30
7.1 7.2
7.2
±
Compost suspension 1% (v/v) Compost suspension 2% (v/v) Compost suspension 1.5% (v/v) Compost suspension 3% (v/v)
Plateau phase not reached.
Table 4 ISO 14852 (1999) ± Avicel (reference substance) Laboratory
Degree of degradation (% ThCO2 )
Degradation based on Cbalance (%)
Input material (mg/l)
Test duration (d)
Temperature (°C)
pHend with test material
pHend blank
Inoculum
1 4 6
64a 73 53
±
77 75
1000 2250 100
35 50 49
25 35 20
7.0 ± 7.1
7.0 ± 7: 0
7
50 92
88 95
100
46
23
7.1 7.0
6.9 7.0
Compost suspension Compost suspension Compost suspension 3% (v/v)
84 88 81b
84 86
100
42
25
±
±
100
28
22
7.3
7.3
±
200
50
23
104a
100
56
±
7.1 7.1 ±
7.1 7.2 ±
8 9 10 11 a b
79b 76b 92
±
Compost suspension 2% (v/v) Activated sludge (30 mg/l MLSS) 10 mg/l MLSS activated sludge Activated sludge Activated sludge (30 mg/l MLSS)
Average of multiple measurements. Plateau phase not reached.
the degradation curves show no lag phases. In some cases up to 40% of the polymer carbon had been transferred into biomass; here the degree of degradation if calculated solely from the oxygen demand, would not re¯ect the degradability of the material, which underlines the value of a carbon balance. Compared to the test material Mater-Bi ZI01U a signi®cant slower degradation was observed for the copolyester ABA 618. The degrees of biodegradation were usually at 3±31% based on ThOD and 4±40% including carbon balance (Table 3, Fig. 5), only in one case a signi®cantly higher biodegradability of 77% BOD of
ThOD and 98% with carbon balance was observed. This is probably attributable to the inoculum because in this case activated sludge was used instead of a compost suspension. In most cases the tests were terminated before a clear plateau phase was reached and the test material which is known to be inherently biodegradable was not completely degraded under the conditions of this test. As a consequence of these results the test duration was extended up to six months and it may be expected that under these conditions a higher degree of biodegradation can be reached as demonstrated with a comparable test material in the BASF laboratory.
U. Pagga et al. / Chemosphere 42 (2001) 319±331
325
Table 5 ISO 14852 (1999) ± Mater-Bi ZI01U Degree of degradation (% ThCO2 )
Degradation based on Cbalance (%)
Input material (mg/l)
Test duration (d)
Temperature (°C)
pHend with test material
pHend blank
Inoculum
1 3
67a 80
± 94
1000 400
35 33
25 20
7.0 7: 0
7.0 7: 0
4 6
83 57 14
98 65 44
2250 100
50 49
35 20
7.0 ± 6.8
7.0 ± 7.0
Compost suspension Activated sludge 1% (v/v)
28 15 73
100
46
23
7.0 7.0 6.98
7.0
7
6 6 64
7.0
8
81
81
100
42
25
±
±
9
85
91
100
28
22
7.3
7.3
87 54b 48b 86
94 ±
200
50
23
94a
100
56
±
7.13 7.10 ±
7.13 7.16 ±
Laboratory
10 11 a b
Compost suspension Compost suspension 3% (v/v) Compost suspension 2% (v/v) Activated sludge (30 mg/l MLSS) 10 mg/l MLSS activated sludge Activated sludge Activated sludge (30 mg/l MLSS)
Average of multiple measurements. Plateau phase not reached.
Table 6 ISO 14852 (1999) ± ABA 618 Laboratory
Degree of degradation (% ThCO2 )
Degradation based on Cbalance (%)
Input material (mg/l)
Test duration (d)
Temperature (°C)
pHend with test material
pHend blank
Inoculum
1 3
15 9
± 16
1000 500
35 40
25 20
7.0 7.3
7.0 7.0
4 6
7 15 5
16 19 16
2250 100
50 49
35 20
7.1 ± 6.9
7.0 ± 7.0
Compost suspension Compost suspension 1% (v/v)
14 13 43
100
46
23
7.0 7.0 7.0
6.9
7
0 0 42
7.0
8
15
13
100
42
25
±
±
9
84 84 9 16
84 85 10 18
100
28
22
7.0
7.3
±
200
50
23
1022
100
56
±
7.0 7.1 ±
7.1 7.1 ±
10 11
30 33 84
Compost suspension Compost suspension 3% (v/v) Compost suspension 2% (v/v) Activated sludge (30 mg/l MLSS)
10 mg/l MLSS sewage sludge sewage sludge Activated sludge (30 mg/l MLSS)
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U. Pagga et al. / Chemosphere 42 (2001) 319±331
Table 7 Details of carbon balances Laboratory
Test material
CCO2 (% of theory)
Cbiomass (% of theory)
CDOC (% of theory)
Cresidual polymer (% of theory)
Ctotal (% of theory)
4
Avicel Mater-Bi ABA 618 ABA 618
92 64 42 14.6 84.3 84.4 9.2
3 9 1 )2.0 )0.6 0.6 0.6
0 0 0 0.3 0.8 1.3 3.8
0 7 18 80 7.3 0.6 63.2
95 80 61 93 92 87 77
8
Fig. 3. ISO 14851 (1999) ± Avicel.
Fig. 4. ISO 14851 (1999) ± Mater Bi Z101U.
U. Pagga et al. / Chemosphere 42 (2001) 319±331
327
Fig. 5. ISO 14851 (1999) ± ABA 618.
Fig. 6. ISO 14852 (1999) ± Avicel.
4.2. ISO 14852 ± CO2 evolution test The valid degrees of biodegradation of Avicel (reference substance) were 64±92% based on ThCO2 and 77±110% including carbon balance (Table 4, Fig. 6). In all but one case a values of biodegradability of >60% were reached. The reason for the low degradation in laboratory 6 was probably that a suspension of active compost was used which was transferred from its temperature optimum of about 60°C to an aquatic environment and a test temperature of 20°C. The degrees of biodegradation, except laboratory 6, of Mater-Bi ZI01U were 48±86% based on ThCO2 and
80±99% including carbon balance (Table 5, Fig. 7). The test results showed in most cases a comparable degradation and only in laboratory 6 for the same reasons as mentioned for Avicel a lower biodegradability. The degrees of biodegradation of ABA 618, exclusive laboratory 6, were 7±84% based on ThCO2 and 16±106% including carbon balance (Table 6, Fig. 8). In this case not only in laboratory 6 but in other laboratories too the test material was found to have a rather low biodegradability. This corresponds to the results of the respirometric test. In two cases a rather high degree of biodegradation, compared to other experiments, was observed which is probably again attributable to the use
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U. Pagga et al. / Chemosphere 42 (2001) 319±331
Fig. 7. ISO 14852 (1999) ± Mater Bi Z101U.
Fig. 8. ISO 14852 (1999) ± ABA 618.
of activated sludge as inoculum. In this test the results of the carbon balance do not in some cases correspond to the values based on CO2 measurement. The option to use a carbon balance is shown for two laboratories in more detail in Table 7. It includes not only the determination of the carbon of the CO2 evolved and the carbon in the new biomass, as shown in Tables 1±6, but also the water-soluble DOC and the carbon of the residual test material. A complete carbon balance provides information about the accuracy of the test, when the sum of total carbon is at least at about 90%. Lower ®gures, as it is the case in some of the examples shown in Table 7 may probably be due the problem of correct biomass or the residual polymer determination.
5. Discussion All 11 participating laboratories were asked to give their comments and report their experience. As both test methods are based on well known and often used standard methods (ISO 9408, 1999; ISO 9439, 1999) some of the laboratories already had experience with this type of biodegradation testing. These basic methods have shown their suitability for many dierent test materials for many years and are often used to determine biodegradability of chemicals. During the development of the German standard DIN V 54900, parts of which are identical to ISO 14851 (1999) and ISO 14852 (1999), practical experience with the modi®ed new test varia-
U. Pagga et al. / Chemosphere 42 (2001) 319±331
tions was gained as well and meanwhile both methods have been standardized and are used. So it can be concluded that based on the ring test sucient experience is available with both aquatic tests for polymer degradation and one major goal of the ring test has been achieved. Results of biological and especially of biodegradability tests usually have a much higher variation than chemical analyses and too high reproducibility of such test results cannot be expected. Experience shows that even in the same laboratory using the same test conditions signi®cantly dierent results can be obtained. Especially for the standards under investigation several test conditions are generally possible and were varied in a wide range in this ring test, e.g., inoculum, temperature or test material concentration. Therefore a relatively high deviation of the results is not surprising and can be accepted. The variability of test conditions is sensible for practical reasons because the aim of these tests is not the simulation of an aquatic environment but ®rst and foremost a possibility of obtaining information on the ultimate biodegradability of polymeric test materials, e.g., as one prerequisite for an evaluation of their compostability. One of the most important factors for the variability, the in¯uence of the inoculum on the test results, cannot be predicted and standardized but only described by the quality criteria used. Based on this background the test data in this ring test in most cases were in sucient agreement and the observed deviations can be explained. The reason for the low degradability measured in one laboratory in contrast to others was probably, as already mentioned, the transfer of a compost from a terrestrial environment and a temperature optimum of about 60°C to an aquatic test system and the lower temperature conditions of about 20°C. The in¯uence of the temperature change could be demonstrated in this laboratory using Avicel and a polyester which was chemically nearly identical with ABA 618. Both materials showed a biodegradation >60% when tested at 58°C in aquatic tests. It is therefore very important to chose an optimal inoculum or to adjust the inoculum to the test temperature carefully by an adaptation period. In some respirometers a temperature higher than 30°C may be technically problematic as the equipment may suer from too much humidity in the water bath whereas in the CO2 evolution test a temperature of up to 60°C gives usually less problems and needs only some few technical modi®cations of the usual test design. Laboratories which used activated sludge as inoculum generally obtained a better degradability than in the case of compost suspensions. This can be explained with a higher degradation potential of activated sludge in an aquatic test and the stress for the microorganisms when they are transferred from a terrestrial to an aquatic environment.
329
The test results and the experience also con®rm that the test methods are suitable even when the test material is not water-soluble. Biochemical oxygen demand and carbon conversion are unequivocal indicators of biodegradation processes and can be measured precisely and reproducibly even for poorly water-soluble materials. Measurement of the exhaust gas requires tight connections and a suciently exact determination of the CO2 which are no problems. The disadvantage of the usual analytical method, absorption of CO2 in sodium hydroxide solution and the determination of the inorganic carbon (IC) content of the absorber solution in a carbon analyzer, is the frequent handling of concentrated alkaline solutions and the possibility of losing CO2 if the absorption capacity of the solution is not sucient. But the analytical techniques of this test may be improved in future to get for example a continuous measurement of CO2 by conductivity. Also online measurements by infrared absorption could avoid the disadvantage of discontinuous measurements but they can run into other diculties because this technique needs an exact measurement of the gas ¯ow. For low degradation rates the CO2 concentrations becomes small and the gas ¯ow in such a test system is generally very low. The advantage of the CO2 test is that it can better be run at temperatures considerably higher than 20°C than the respirometric test. Both tests may be improved in future as there is a considerable potential for automation and the handling of the test data by computer, which signi®cantly improves their performance and evaluation. The test duration of about 50 days, applied from most laboratories participating on this ring test, has proved to be too short in some cases for the evaluation of degradability of polymeric materials. In some of the tests the plateau phase had not yet been reached when the test was ®nished. The experience of the ring test and with the routinely used tests has shown that it is possible to keep the aquatic tests to a longer test period, although in both tests, gases are measured (oxygen or CO2 ) and the complete test systems must be kept airtight continuously over the entire period. The biological systems have also sucient stability and activity, especially in the highly buered medium, over longer periods. An extension of the test duration up to six months is therefore possible and should be applied if necessary. One experience obtained in the ring test was the fact that a carbon balance may be a helpful and suitable tool in determining the complete degradability of a test material. Both, the carbon evolved as CO2 and transferred to new biomass can be regarded as biodegraded and contribute to the correct calculation of the degree of biodegradation. It is, however, not yet possible to present a generally accepted method in the standards but just to give examples. Important prerequisites are a
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suitable analytical method for determining the residual test material and the increase in biomass of the inoculum. For the ®rst, usually analytical techniques such as gel chromatography will be used, for the latter, the determination of protein is suitable (Sperandio and P uchner, 1993; Urstadt et al., 1995b). These restrictions may also be the reason that in some cases higher degrees of degradation than 100% were determined. A general experience is that the test material in laboratory tests should be as ®ne a powder as possible to improve the bioavailability. Problems could, however, arise with hydrophobic test materials, which could stick to the wall of the test vessel, for example, and could therefore be less available. Generally it can be stated, that with both standards used in this ring test, ISO 14851 (1999) and 14852 (1999) biodegradation of polymeric materials can be determined with sucient accuracy and do not need more exact speci®cations as this type of method requires a certain freedom in application. The dierences in degradation degrees for the two test materials and the reference substance under investigation were reproduced by most experiments. The deviations of some test results indicate, however, the limits of aquatic tests for polymeric materials. These tests are not designed to simulate distinct environments, such as rivers or waste water treatment plants but to evaluate the principle biodegradability using the already mentioned advantages of aquatic test systems. For this purpose an optimisation of the original methods ISO 9408 (1999) and ISO 9439 (1999) was necessary and useful but nevertheless principle restrictions of aquatic tests for polymers are left, for example the better growth conditions of actinomycetes and fungi which are important polymer degrading micro-organisms in terrestrial systems. Therefore the more powerful but also much more expensive aerobic composting test ISO 14855 (1999) can be an alternative. For polymeric materials in this test higher degradation rates and degrees have been observed as in the aquatic tests at room temperature. Knowing these general restrictions the results of the ring test have shown that both aquatic tests are suitable test methods for determining the biodegradability of polymeric plastic materials in water. Acknowledgements On behalf of IBRG and ISO the authors would like to thank the co-operating laboratories for their contributions. Fraunhofergesellschaft Freising, Germany (M. Menner); Kuraray Co. Ltd., Kurashiki, Japan (M. Shimamura); University of Stuttgart, Germany (A. Sch afer); Institute for Agrobiotechnology, Tulln, Austria (U. Link); Bayer AG, Leverkusen, Germany (G. M uller); BASF AG, Ludwigshafen, Germany
(D. Beimborn and U. Pagga); CTP, Grenoble, France (Y. Aitkin); CITI, Kurume, Japan (Y. Yakabe); OWS Ghent, Belgium (B. de Wilde); Swedish National Testing and Research Institute, Boras, Sweden (C. Lindblad); Hydrotox GmbH, Freiburg, Germany (S. Gartiser).
References Deutsches Institut f ur Normung, 1998. Testing of the compostability of polymeric materials ± German standard DIN V 54900 parts 1±3. European Standard EN 13432, (Draft 1999). Requirements for packaging recoverable through composting and biodegradation ± Test scheme and evaluation criteria for the ®nal acceptance of packaging. International Standard 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. International Standard ISO 9439, 1999. Water quality ± Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium ± Carbon dioxide evolution test. International Standard ISO 14851, 1999. Plastics ± Determination of the ultimate aerobic biodegradability of plastic materials in an aqueous medium ± Method by determining the oxygen demand in a closed respirometer. International Standard ISO 14852, 1999. Plastics ± Determination of the ultimate aerobic biodegradability of plastic materials in an aqueous medium ± Method by analysis of evolved carbon dioxide. International Standard ISO 14855, 1999. Plastics ± Determination of the ultimate aerobic biodegradability and disintegration of plastic materials under controlled composting conditions ± Method by analysis of evolved carbon dioxide. International Standard ISO TR 15462, 1997. Water quality ± Selection of tests for biodegradability. International Standard Committee Draft 15986. Plastics ± Evaluation of compostability ± Test scheme for ®nal acceptance. OECD Guidelines for testing of chemicals, 1993. Paris 301 B CO2 Evolution test and 301 F Manometric respirometry test. Pagga, U., Beimborn, D.B., Boelens, J., DeWilde, B., 1995. Determination of the aerobic biodegradability of polymeric material in a laboratory controlled composting test. Chemosphere 31, 4475±4487. Pagga, U., Beimborn, D.B., Yamamoto, M., 1996. Biodegradability and compostability of polymers ± test methods and criteria for evaluation. J. Environ. Polymer Degr. 4, 173± 177. Pagga, U., 1997a. Compostability and biodegradability of plastic materials: a new quality of materials properties. Mater. Res. Soc. MRS Bulletin 22 (11), 5±7. Pagga, U., 1997b. Testing biodegradability with standardized methods. Chemosphere 35, 2953±2972. Pagga, U., 1998. Biodegradability and compostability of polymeric materials in the context of the European pack-
U. Pagga et al. / Chemosphere 42 (2001) 319±331 aging regulation. Polymer Degradation and Stability 59, 371±376. Pagga, U., 1999. Compostable packaging materials ± test methods and limit values for biodegradation. Appl. Microbiol. Biotechnol. 51, 125±133. P uchner, P., Mueller, W.R., Bardtke, D., 1995. Assessing the biodegradation potential of polymers in screening and longterm test systems. J. Environ. Polymer Degr. 3, 133±143. Schafer, A., 1999. Beurteilung der biologischen Abbaubarkeit ber Sauerstoverbrauch, unter aeroben Bedingungen u Kohlenstodioxidproduktion und Kohlenstobilanz, Stuttgarter Berichte zur Abfallwirtschaft Band 72, Erich Schmidt: Verlag.
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Sperandio, A., P uchner, P., 1993. Bestimmung der gesamtproteine als biomasse-parameter in w assrigen kulturen und auf tr agermaterialien aus bioreaktoren-modi®zierte methode nach lowry±eine praktikable methode in der umweltanalytik. Wasser Abwasser 134 482±485. Urstadt, S., Augusta, J., M uller, R.J., Deckwer, W.D., 1995a. Calculation of carbon balances for evaluation of the biodegradability of polymers. J. Environ. Polymer Degr. 3, 121±131. Urstadt, S., Augusta, J., M uller, R.J., Deckwer, W.D., 1995b. Calculation of carbon balances for evaluation of the biodegradability of polymers. J. Environ. Polymer Degr. 3, 121±131.
Determination of the aerobic biodegradability of polymeric material in aquatic batch tests Udo Pagga
a,*
, Anja Sch afer b, Rolf-Joachim M uller c, Michael Pantke
d
a
BASF Aktiengesellschaft Ecology, D-67056 Ludwigshafen, Germany Endress und Hauser, Dieselstr. 24, D-10839 Gerlingen, Germany (formerly University Stuttgart) Gesellschaft f ur Biotechnologische Forschung mbH Mascheroder Weg 1, D-38124 Braunschweig, Germany d Bundesanstalt f ur Materialpr ufung Unter den Eichen 87, D-12205 Berlin, Germany b
c
Received 27 September 1999; accepted 8 February 2000
Abstract Results of an international ring-test of two laboratory methods are presented for investigating the biodegradability of organic polymeric test materials in aquatic test systems based on respirometry and the evolution of carbon dioxide. These methods are developed further from the well-known standardized biodegradation tests ISO 9408 (1999) and ISO 9439 (1999), which have been successfully used for many years. The most important improvements are the extension of the test period up to six months, the increase of the buer capacity and nutrient supply of the inorganic medium, an optimization of the inoculation, and optionally, the possibility of a carbon balance. A ring test, organized by the International Biodeterioration Research Group (IBRG), was run using a poly(,-caprolactone)-starch blend and an aliphatic-aromatic co-polyester as test materials and a microcrystalline cellulose powder as a reference material. The test results and the experience gained by the participants showed that the methods are suitable and practicable. The test methods have been meanwhile established as standards ISO 14851 (1999) and ISO 14852 (1999). Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Aerobic biodegradability; Respirometry; Carbon dioxide evolution; Polymeric materials; Cellulose powder
1. Introduction Composting of solid waste has been recognised as a valuable method for waste treatment. Typical input materials are not only green waste from gardens, biowaste from households and natural materials such as paper, but also manufactured products, for example waste bags, which are more and more based on biodegradable plastics to allow this biological waste
*
Corresponding author. Tel.: 621-60-58148; fax: 621-6058043. E-mail address: [email protected] (U. Pagga).
treatment. Compostable products, especially synthetic plastic materials, need to be identi®ed and classi®ed as being biodegradable and must not adversely in¯uence the composting process or the quality of the compost produced (Pagga et al., 1996; Pagga, 1997a, 1998). Test methods and test strategies for this purpose have been developed and recently standardized on national (e.g., in Germany DIN 54900, 1998) and European level (EN 13432 (Draft 1999)) and worldwide (ISO, 15986). As biodegradability is a basic prerequisite for chemicals in general a number of biodegradation tests have been developed and standardized in the last 25 years (ISO 15462, 1997; Pagga, 1997b). Most of them are intended to determine aerobic biodegradability of
0045-6535/01/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 0 ) 0 0 0 6 9 - 2
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organic chemicals in aquatic batch systems such as the well-known and much-used manometric respirometry test (OECD 301 F, 1993; ISO 9408, 1999), the twophase closed bottle test (ISO 10708) or the CO2 evolution test (OECD 301 B, 1993; ISO 9439, 1999). But only recently have special tests for polymeric materials been suggested and standardized. One is the aerobic composting test (Pagga et al., 1995; ISO 14855, 1999); further possibilities are specialised aquatic screening tests. The usual aquatic biodegradation tests have a considerably lower degradation potential than terrestrial tests, because fungi and actinomycetes, which are very important for polymer degradation, do not have optimum growth conditions in water. The standard test duration of 28 days applied in such tests is also too short for polymeric test materials. Therefore the respirometric test (P uchner et al., 1995) and the CO2 evolution test (Urstadt et al., 1995a; Sch afer, 1999) have been modi®ed and improved for this speci®c application by using, for example, suspensions of compost as inoculum, inorganic media with higher buering capacity and more nutrients, and by the extending of the test duration and the temperature range. The advantage of the aquatic tests is that they have a somewhat lower requirement of resources for the study and they can be used to prepare a carbon balance which characterises the extent of biodegradation better than the CO2 evolution alone, particularly if a signi®cant amount of biomass is formed during the test. A carbon balance is not possible in a terrestrial test using compost because there is too much organic carbon in the compost. The use of the synthetic organic carbon-free mineral medium in the aquatic test avoids the so-called priming eect, a stimulation of the degradation by addition of plastic samples to a compost which cannot be corrected by the blank values. These aquatic tests provide, however, in the case of plastic material in ®rst line basic information on biodegradation and do not have the character of a simulation test for an aquatic environment. The biodegradation in a solid compost matrix at temperatures up to 60°C may be better simulated in the aerobic composting test (Pagga et al., 1995; ISO 14855, 1999). The Plastic Committee TC61 of the International Standardization Organization (ISO) decided to standardize these aquatic tests and working group 22 of ISO TC 61/SC 5 had the task of preparing the International Standards. In the course of standardization a ring test was performed with the principal aim of investigating the suitability of the methods for testing plastic materials and of allowing a number of laboratories to obtain more practical experience. The test was organized by the Biodegradable Plastics Group of the International Biodeterioration Research Group (IBRG). This publication describes the test methods and presents the results of the participants of the ring test.
2. Test methods The International Standards ISO 14851 (1999) and ISO 14852 (1999) are specially designed for determining the aerobic biodegradability of polymeric and plastic materials including formulation additives. In both tests the test material is exposed under laboratory conditions in an aqueous medium to an inoculum with a wide variety of microorganisms which is either activated sludge or a compost suspension. According to the test procedure activated sludge is collected from a well-operated sewage treatment plant or a laboratory plant and used in a concentration range of 30±1000 mg/l suspended solids in the ®nal test mixture. When biodegradation processes in a natural aquatic environment should be simulated or a carbon balance is carried out, an inoculum concentration of 30 mg/l is recommended. In the case of a compost inoculum 10 g of non-sterile compost from a composting plant treating predominantly organic waste are suspended in 100 ml of inorganic test medium. The supernatant is used after settling or ®ltration at a recommended concentration of 1±5% (v/v) in the test ¯asks. The use of compost can increase the number of fungi in the test ¯asks and improve the biodegradation of plastic materials. Instead of compost fertile soil may be used as well. The inorganic salts of the medium can optionally be used in a concentration about ten-fold higher than the usual standardized biodegradation tests (see ISO 15462, 1997). This is to improve the buer capacity of the medium and the available amount of inorganic nutrients especially nitrogen and phosphorus salts. The test duration is extended to up to six months compared to usually four weeks in standard aquatic biodegradation tests. The temperature may be chosen in a broad range to allow especially the growth of thermophilic microorganisms which may have a better degradative potential for polymers than mesophilics. ISO 14851 (1999) speci®es a method by determining the biochemical oxygen demand (BOD) in a closed respirometer. The test mixture of test material, inorganic medium and inoculum is stirred in closed ¯asks of the respirometer (Fig. 1). Evolved carbon dioxide (CO2 ) is absorbed in a suitable absorber (e.g., soda lime) in the head space of the test ¯asks. The oxygen consumption is determined by measuring the amount of oxygen required to maintain a constant gas volume in the respirometer ¯ask, or by measuring the change in volume or pressure either automatically or manually. The level of biodegradation is determined by comparing the BOD with the theoretical oxygen demand (ThOD) and expressed in percent. The in¯uence of possible nitri®cation processes on the BOD has to be considered in cases of polymers containing nitrogen. A variation of this test which needs no expensive respirometer is described in annex D of ISO 14851
U. Pagga et al. / Chemosphere 42 (2001) 319±331
321
Fig. 1. Principle of a closed respirometer ± ISO 14851 (1999) (example).
(1999). The method is identical to the two-phase closed bottle test ISO 10708. The inoculated test mixture is shaken or stirred in the test bottles. Known volumes of medium and air are used to assure steady-state oxygen partitioning between liquid and gas phases. The degradation is followed by regularly measuring the dissolved oxygen concentration in the aqueous phase usually by an oxygen electrode. The oxygen uptake is calculated from the measured dissolved oxygen concentrations divided by the oxygen saturation value under normal conditions and multiplied by the total oxygen content originally present in the liquid and gaseous phases. Biodegradability is then calculated and expressed as in the case of the respirometer. The second aquatic test, ISO 14852 (1999) is based on the determination of the evolved CO2 (Fig. 2). The same test mixture as for ISO 14851 (1999) is agitated in test ¯asks and aerated with CO2 -free air over the test period.
The CO2 evolved during the microbial degradation which is in the exhaust air is determined by suitable methods. Such methods are, for example, absorption of CO2 in sodium hydroxide solution and determination of dissolved inorganic carbon (DIC) in a carbon analyzer, absorption of CO2 in a barium hydroxide solution and subsequent titration, gas chromatography or continuous infrared detection. The level of biodegradation is calculated by comparing the evolved CO2 with the theoretical amount (ThCO2 ) and again expressed in percent. The process of biodegradation is shown in both tests in a curve where either BOD or CO2 production or the calculated biodegradation percentage is plotted as a function of time. Typical biodegradation curves consist of a lag phase, which is the time from the start of a test until a distinct biodegradation (e.g., 10%) can be measured, a biodegradation phase, in which the maximum degradation takes place and a plateau phase, in which it
Fig. 2. Principle of the CO2 evolution test ± ISO 14852 (1999) (example).
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is almost completed. The test result is the biodegradation degree in percent, calculated as a mean value from the measured data of the plateau phases of parallel test vessels. The methods described here are used to determine the ultimate biodegradation which is the complete utilisation of a test material by microorganisms resulting in the production of carbon dioxide, water, mineral salts and new microbial cellular constituents (biomass) in contrast to a primary biodegradation which is only the change of the identity of the test material (Pagga, 1999). As an option a carbon balance may be performed and calculated to obtain additional information on biodegradation. Plastic materials are normally of more complex composition than substances with low molecular weights. Therefore determination of CO2 evolution or of BOD alone may not be sucient to characterize and quantify their biodegradability. During biodegradation new biomass is built up by the microorganisms and some of the organic carbon of the test material used is transformed to biomass but not biochemically oxidised. That is the reason why the analytical parameter CO2 evolution and BOD will often not reach 100% of the theoretical values ThCO2 and ThOD even in the case of complete biodegradation of a test material and insucient degradation could falsely be assessed from the test results. A balance of the fate of the organic carbon of the test material may be helpful to con®rm complete biodegradability. The principle of such a balance is the detailed quantitative measurement of the CO2 evolved (either directly determined in the exhaust air or calculated from the measured BOD), the new produced biomass (determined, e.g., as protein), the water soluble metabolites generated during the biodegradation process (determined as dissolved organic carbon DOC), and the residual undegraded polymeric test material using preferably a substance speci®c analytical method. The measured values are transformed to carbon and the sum is compared with the amount of the organic carbon of the test material introduced into the test system. As an option a carbon balance may be performed and calculated to obtain additional information on biodegradation. Plastic materials are normally of more complex composition than substances with low molecular weights. Therefore the determination of CO2 evolution or of BOD alone may not be sucient to characterize and quantify their biodegradability. During biodegradation new biomass is built up by the microorganisms and some of the organic carbon of the test material used is transformed to biomass but not biochemically oxidised. Therefore analytical parameters such as CO2 evolution and BOD will often not reach 100% of the respective theoretical values even in the case of complete biodegradation of a test material and an insucient degradation could falsely be evaluated from the test results. A balance of the fate of the organic carbon of the test material may be helpful to con®rm
complete biodegradability. The principle of such a balance is the detailed quantitative measurement of the CO2 evolved (either directly determined in the exhaust air or calculated from the measured BOD), the new produced biomass, the water soluble metabolites generated during the biodegradation process (determined as dissolved organic carbon DOC), and the residual undegraded polymeric test material. The measured values are transformed to carbon and the sum is compared with the amount of the organic carbon of the test material introduced at the beginning into the test system. Two steps of the carbon balance are crucial, the determination of the biomass and the residual polymers. Biomass may be determined at the beginning and the end of the test in the inoculum using suitable methods, e.g., by protein measurement. From the measured value the amount of carbon in the biomass is determined. From the dierence the increase of organic carbon of the biomass is calculated. The residual polymers can be extracted at the end of the test in the total remaining samples and directly determined if any polymer speci®c analysis is available. From the usually known composition of the polymers the amount of carbon can be calculated. If no suitable analytical technique is available also an indirect determination is possible, e.g., by washing, drying and weighing the residue and determining the total organic carbon (TOC). From this TOC the organic carbon of the biomass is subtracted to obtain the amount of carbon of the residual polymers. 3. Test materials Mater Bi ZI01U a poly(,-caprolactone)-starch blend obtained from Novamont S.P.A., Novara, Italy, with a mean particle size of <180 lm and the following elemental composition: carbon 53.6%; hydrogen 8.0%; nitrogen 0%; oxygen 38.4% (w/w) as stated by the supplier. ABA 618 an aliphatic-aromatic copolyester with the following elemental composition: carbon 61.7%; hydrogen 8.8%; nitrogen 5.7%; oxygen 23.8% (w/w) and with a mean molar mass of a monomer of 147.8 as stated by the supplier. As a positive control for the check of the activity of the inoculum during the test a microcrystalline cellulose powder, Avicel, was used with the following elemental composition: carbon 41.8%; hydrogen 6.4%; nitrogen 0%; oxygen 52.0% (w/w) as measured by BASF. Avicel (Nr. 2330) was obtained from Merck, Darmstadt, Germany. 4. Results The results of the ring test are shown in Tables 1±7 and Figs. 3±8. The carbon balance is just based on BOD
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323
Table 1 ISO 14851 (1999) ± Avicel (reference substance) Laboratory
Degree of degradation (% ThOD)
Degradation based on Cbalance (%)
Input material (mg/l)
Test duration (d)
Temperature (°C)
pHend with test material
pHend blank
Inoculum
1 2
69 79
± ±
± 100
28 28
25 25
6.8 7.4
7.6 7.5
3
84
100
500
40
20
7: 0
7: 0
4
82 67
90 86
160
41
35
7.0 9.6
7.0 8.6
Compost suspension Activated sludge (30 mg/l MLSS) Activated sludge 1% (v/v)
5
79
100
62
20
±
±
6
61 61 73
67
100
47
20
7.2
7.2
70
65
7.2
7.2
±
Compost suspension 2% (v/v) Compost suspension 1.5% (v/v) Compost suspension 3% (v/v)
Table 2 ISO 14851 (1999) ± Mater-Bi ZI01U
a
Laboratory
Degree of degradation (% ThOD)
Degradation based on Cbalance (%)
Input material (mg/l)
Test duration (d)
Temperature (°C)
pHend with test material
pHend blank
Inoculum
1 3
63a 76
±
82
1000 400
28 36
25 20
7.1 7.0
7.6 7.0
4
80
93
160
41
35
8.7
8.6
5
50
100
62
20
±
±
6
44 59 60
Compost suspension Activated sludge 1% (v/v) Compost suspension 2% (v/v) Compost suspension 1.5% (v/v)
102
100
47
20
7: 2
7: 2
47 60
83 103
7.2 7.2
7.2
±
Compost suspension 3% (v/v)
Plateau phase not reached.
or CO2 evolution and the determination of biomass. The source and concentration of the inoculum is indicated as given by the participating laboratories (MLSS mixed liquor suspended solids). 4.1. ISO 14851 respirometric test The degrees of biodegradation of the reference substance Avicel were 61±84% based on ThOD and 65± 100% including carbon balance (Table 1, Fig. 3). The course of the degradation curves are comparable, particularly taking into account the dierent test temperatures and the dierent inocula used. The lag phases in all tests were coherently around ®ve days. Dierences increased with progressing degradation, however, all
degrees of degradation at the end of the tests exceeded the limit value of 60% which is requested by the standard to prove the validity of the test. Some tests were ®nished before a clear plateau was reached, in these cases higher ®nal degrees of degradation could be expected. With the exception of one laboratory, the carbon found in the biomass as one part of the carbon balance contributed signi®cantly to the degree of biodegradation. The degrees of biodegradation of Mater Bi ZI01U were 44±80% based on ThOD and 82±103% including carbon balance (Table 2, Fig. 4). The variation between the degradation curves is greater than in the case of Avicel. The observed degrees of degradation are comparable with those found with the positive control, but
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Table 3 ISO 14851 (1999) ± ABA 618
a
Laboratory
Degree of degradation (% ThOD)
Degradation based on Cbalance (%)
Input material (mg/l)
Test duration (d)
Temperature (°C)
pHend with test material
pHend blank
Inoculum
1 2
4 77a
±
98
1000 100
28 28
25 25
7.7 7.2
7.6 7.2
3
7 14
104 22
500
40
20
7.4 7: 0
7.4 7: 3
Compost suspension Activated sludge (30 mg/l MLSS)
4
31
40
160
41
35
8: 6
8: 7
5
23
100
62
20
±
±
6
11 7
11
100
47
20
7.1
7.2
3 17
4 30
7.1 7.2
7.2
±
Compost suspension 1% (v/v) Compost suspension 2% (v/v) Compost suspension 1.5% (v/v) Compost suspension 3% (v/v)
Plateau phase not reached.
Table 4 ISO 14852 (1999) ± Avicel (reference substance) Laboratory
Degree of degradation (% ThCO2 )
Degradation based on Cbalance (%)
Input material (mg/l)
Test duration (d)
Temperature (°C)
pHend with test material
pHend blank
Inoculum
1 4 6
64a 73 53
±
77 75
1000 2250 100
35 50 49
25 35 20
7.0 ± 7.1
7.0 ± 7: 0
7
50 92
88 95
100
46
23
7.1 7.0
6.9 7.0
Compost suspension Compost suspension Compost suspension 3% (v/v)
84 88 81b
84 86
100
42
25
±
±
100
28
22
7.3
7.3
±
200
50
23
104a
100
56
±
7.1 7.1 ±
7.1 7.2 ±
8 9 10 11 a b
79b 76b 92
±
Compost suspension 2% (v/v) Activated sludge (30 mg/l MLSS) 10 mg/l MLSS activated sludge Activated sludge Activated sludge (30 mg/l MLSS)
Average of multiple measurements. Plateau phase not reached.
the degradation curves show no lag phases. In some cases up to 40% of the polymer carbon had been transferred into biomass; here the degree of degradation if calculated solely from the oxygen demand, would not re¯ect the degradability of the material, which underlines the value of a carbon balance. Compared to the test material Mater-Bi ZI01U a signi®cant slower degradation was observed for the copolyester ABA 618. The degrees of biodegradation were usually at 3±31% based on ThOD and 4±40% including carbon balance (Table 3, Fig. 5), only in one case a signi®cantly higher biodegradability of 77% BOD of
ThOD and 98% with carbon balance was observed. This is probably attributable to the inoculum because in this case activated sludge was used instead of a compost suspension. In most cases the tests were terminated before a clear plateau phase was reached and the test material which is known to be inherently biodegradable was not completely degraded under the conditions of this test. As a consequence of these results the test duration was extended up to six months and it may be expected that under these conditions a higher degree of biodegradation can be reached as demonstrated with a comparable test material in the BASF laboratory.
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Table 5 ISO 14852 (1999) ± Mater-Bi ZI01U Degree of degradation (% ThCO2 )
Degradation based on Cbalance (%)
Input material (mg/l)
Test duration (d)
Temperature (°C)
pHend with test material
pHend blank
Inoculum
1 3
67a 80
± 94
1000 400
35 33
25 20
7.0 7: 0
7.0 7: 0
4 6
83 57 14
98 65 44
2250 100
50 49
35 20
7.0 ± 6.8
7.0 ± 7.0
Compost suspension Activated sludge 1% (v/v)
28 15 73
100
46
23
7.0 7.0 6.98
7.0
7
6 6 64
7.0
8
81
81
100
42
25
±
±
9
85
91
100
28
22
7.3
7.3
87 54b 48b 86
94 ±
200
50
23
94a
100
56
±
7.13 7.10 ±
7.13 7.16 ±
Laboratory
10 11 a b
Compost suspension Compost suspension 3% (v/v) Compost suspension 2% (v/v) Activated sludge (30 mg/l MLSS) 10 mg/l MLSS activated sludge Activated sludge Activated sludge (30 mg/l MLSS)
Average of multiple measurements. Plateau phase not reached.
Table 6 ISO 14852 (1999) ± ABA 618 Laboratory
Degree of degradation (% ThCO2 )
Degradation based on Cbalance (%)
Input material (mg/l)
Test duration (d)
Temperature (°C)
pHend with test material
pHend blank
Inoculum
1 3
15 9
± 16
1000 500
35 40
25 20
7.0 7.3
7.0 7.0
4 6
7 15 5
16 19 16
2250 100
50 49
35 20
7.1 ± 6.9
7.0 ± 7.0
Compost suspension Compost suspension 1% (v/v)
14 13 43
100
46
23
7.0 7.0 7.0
6.9
7
0 0 42
7.0
8
15
13
100
42
25
±
±
9
84 84 9 16
84 85 10 18
100
28
22
7.0
7.3
±
200
50
23
1022
100
56
±
7.0 7.1 ±
7.1 7.1 ±
10 11
30 33 84
Compost suspension Compost suspension 3% (v/v) Compost suspension 2% (v/v) Activated sludge (30 mg/l MLSS)
10 mg/l MLSS sewage sludge sewage sludge Activated sludge (30 mg/l MLSS)
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U. Pagga et al. / Chemosphere 42 (2001) 319±331
Table 7 Details of carbon balances Laboratory
Test material
CCO2 (% of theory)
Cbiomass (% of theory)
CDOC (% of theory)
Cresidual polymer (% of theory)
Ctotal (% of theory)
4
Avicel Mater-Bi ABA 618 ABA 618
92 64 42 14.6 84.3 84.4 9.2
3 9 1 )2.0 )0.6 0.6 0.6
0 0 0 0.3 0.8 1.3 3.8
0 7 18 80 7.3 0.6 63.2
95 80 61 93 92 87 77
8
Fig. 3. ISO 14851 (1999) ± Avicel.
Fig. 4. ISO 14851 (1999) ± Mater Bi Z101U.
U. Pagga et al. / Chemosphere 42 (2001) 319±331
327
Fig. 5. ISO 14851 (1999) ± ABA 618.
Fig. 6. ISO 14852 (1999) ± Avicel.
4.2. ISO 14852 ± CO2 evolution test The valid degrees of biodegradation of Avicel (reference substance) were 64±92% based on ThCO2 and 77±110% including carbon balance (Table 4, Fig. 6). In all but one case a values of biodegradability of >60% were reached. The reason for the low degradation in laboratory 6 was probably that a suspension of active compost was used which was transferred from its temperature optimum of about 60°C to an aquatic environment and a test temperature of 20°C. The degrees of biodegradation, except laboratory 6, of Mater-Bi ZI01U were 48±86% based on ThCO2 and
80±99% including carbon balance (Table 5, Fig. 7). The test results showed in most cases a comparable degradation and only in laboratory 6 for the same reasons as mentioned for Avicel a lower biodegradability. The degrees of biodegradation of ABA 618, exclusive laboratory 6, were 7±84% based on ThCO2 and 16±106% including carbon balance (Table 6, Fig. 8). In this case not only in laboratory 6 but in other laboratories too the test material was found to have a rather low biodegradability. This corresponds to the results of the respirometric test. In two cases a rather high degree of biodegradation, compared to other experiments, was observed which is probably again attributable to the use
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U. Pagga et al. / Chemosphere 42 (2001) 319±331
Fig. 7. ISO 14852 (1999) ± Mater Bi Z101U.
Fig. 8. ISO 14852 (1999) ± ABA 618.
of activated sludge as inoculum. In this test the results of the carbon balance do not in some cases correspond to the values based on CO2 measurement. The option to use a carbon balance is shown for two laboratories in more detail in Table 7. It includes not only the determination of the carbon of the CO2 evolved and the carbon in the new biomass, as shown in Tables 1±6, but also the water-soluble DOC and the carbon of the residual test material. A complete carbon balance provides information about the accuracy of the test, when the sum of total carbon is at least at about 90%. Lower ®gures, as it is the case in some of the examples shown in Table 7 may probably be due the problem of correct biomass or the residual polymer determination.
5. Discussion All 11 participating laboratories were asked to give their comments and report their experience. As both test methods are based on well known and often used standard methods (ISO 9408, 1999; ISO 9439, 1999) some of the laboratories already had experience with this type of biodegradation testing. These basic methods have shown their suitability for many dierent test materials for many years and are often used to determine biodegradability of chemicals. During the development of the German standard DIN V 54900, parts of which are identical to ISO 14851 (1999) and ISO 14852 (1999), practical experience with the modi®ed new test varia-
U. Pagga et al. / Chemosphere 42 (2001) 319±331
tions was gained as well and meanwhile both methods have been standardized and are used. So it can be concluded that based on the ring test sucient experience is available with both aquatic tests for polymer degradation and one major goal of the ring test has been achieved. Results of biological and especially of biodegradability tests usually have a much higher variation than chemical analyses and too high reproducibility of such test results cannot be expected. Experience shows that even in the same laboratory using the same test conditions signi®cantly dierent results can be obtained. Especially for the standards under investigation several test conditions are generally possible and were varied in a wide range in this ring test, e.g., inoculum, temperature or test material concentration. Therefore a relatively high deviation of the results is not surprising and can be accepted. The variability of test conditions is sensible for practical reasons because the aim of these tests is not the simulation of an aquatic environment but ®rst and foremost a possibility of obtaining information on the ultimate biodegradability of polymeric test materials, e.g., as one prerequisite for an evaluation of their compostability. One of the most important factors for the variability, the in¯uence of the inoculum on the test results, cannot be predicted and standardized but only described by the quality criteria used. Based on this background the test data in this ring test in most cases were in sucient agreement and the observed deviations can be explained. The reason for the low degradability measured in one laboratory in contrast to others was probably, as already mentioned, the transfer of a compost from a terrestrial environment and a temperature optimum of about 60°C to an aquatic test system and the lower temperature conditions of about 20°C. The in¯uence of the temperature change could be demonstrated in this laboratory using Avicel and a polyester which was chemically nearly identical with ABA 618. Both materials showed a biodegradation >60% when tested at 58°C in aquatic tests. It is therefore very important to chose an optimal inoculum or to adjust the inoculum to the test temperature carefully by an adaptation period. In some respirometers a temperature higher than 30°C may be technically problematic as the equipment may suer from too much humidity in the water bath whereas in the CO2 evolution test a temperature of up to 60°C gives usually less problems and needs only some few technical modi®cations of the usual test design. Laboratories which used activated sludge as inoculum generally obtained a better degradability than in the case of compost suspensions. This can be explained with a higher degradation potential of activated sludge in an aquatic test and the stress for the microorganisms when they are transferred from a terrestrial to an aquatic environment.
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The test results and the experience also con®rm that the test methods are suitable even when the test material is not water-soluble. Biochemical oxygen demand and carbon conversion are unequivocal indicators of biodegradation processes and can be measured precisely and reproducibly even for poorly water-soluble materials. Measurement of the exhaust gas requires tight connections and a suciently exact determination of the CO2 which are no problems. The disadvantage of the usual analytical method, absorption of CO2 in sodium hydroxide solution and the determination of the inorganic carbon (IC) content of the absorber solution in a carbon analyzer, is the frequent handling of concentrated alkaline solutions and the possibility of losing CO2 if the absorption capacity of the solution is not sucient. But the analytical techniques of this test may be improved in future to get for example a continuous measurement of CO2 by conductivity. Also online measurements by infrared absorption could avoid the disadvantage of discontinuous measurements but they can run into other diculties because this technique needs an exact measurement of the gas ¯ow. For low degradation rates the CO2 concentrations becomes small and the gas ¯ow in such a test system is generally very low. The advantage of the CO2 test is that it can better be run at temperatures considerably higher than 20°C than the respirometric test. Both tests may be improved in future as there is a considerable potential for automation and the handling of the test data by computer, which signi®cantly improves their performance and evaluation. The test duration of about 50 days, applied from most laboratories participating on this ring test, has proved to be too short in some cases for the evaluation of degradability of polymeric materials. In some of the tests the plateau phase had not yet been reached when the test was ®nished. The experience of the ring test and with the routinely used tests has shown that it is possible to keep the aquatic tests to a longer test period, although in both tests, gases are measured (oxygen or CO2 ) and the complete test systems must be kept airtight continuously over the entire period. The biological systems have also sucient stability and activity, especially in the highly buered medium, over longer periods. An extension of the test duration up to six months is therefore possible and should be applied if necessary. One experience obtained in the ring test was the fact that a carbon balance may be a helpful and suitable tool in determining the complete degradability of a test material. Both, the carbon evolved as CO2 and transferred to new biomass can be regarded as biodegraded and contribute to the correct calculation of the degree of biodegradation. It is, however, not yet possible to present a generally accepted method in the standards but just to give examples. Important prerequisites are a
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suitable analytical method for determining the residual test material and the increase in biomass of the inoculum. For the ®rst, usually analytical techniques such as gel chromatography will be used, for the latter, the determination of protein is suitable (Sperandio and P uchner, 1993; Urstadt et al., 1995b). These restrictions may also be the reason that in some cases higher degrees of degradation than 100% were determined. A general experience is that the test material in laboratory tests should be as ®ne a powder as possible to improve the bioavailability. Problems could, however, arise with hydrophobic test materials, which could stick to the wall of the test vessel, for example, and could therefore be less available. Generally it can be stated, that with both standards used in this ring test, ISO 14851 (1999) and 14852 (1999) biodegradation of polymeric materials can be determined with sucient accuracy and do not need more exact speci®cations as this type of method requires a certain freedom in application. The dierences in degradation degrees for the two test materials and the reference substance under investigation were reproduced by most experiments. The deviations of some test results indicate, however, the limits of aquatic tests for polymeric materials. These tests are not designed to simulate distinct environments, such as rivers or waste water treatment plants but to evaluate the principle biodegradability using the already mentioned advantages of aquatic test systems. For this purpose an optimisation of the original methods ISO 9408 (1999) and ISO 9439 (1999) was necessary and useful but nevertheless principle restrictions of aquatic tests for polymers are left, for example the better growth conditions of actinomycetes and fungi which are important polymer degrading micro-organisms in terrestrial systems. Therefore the more powerful but also much more expensive aerobic composting test ISO 14855 (1999) can be an alternative. For polymeric materials in this test higher degradation rates and degrees have been observed as in the aquatic tests at room temperature. Knowing these general restrictions the results of the ring test have shown that both aquatic tests are suitable test methods for determining the biodegradability of polymeric plastic materials in water. Acknowledgements On behalf of IBRG and ISO the authors would like to thank the co-operating laboratories for their contributions. Fraunhofergesellschaft Freising, Germany (M. Menner); Kuraray Co. Ltd., Kurashiki, Japan (M. Shimamura); University of Stuttgart, Germany (A. Sch afer); Institute for Agrobiotechnology, Tulln, Austria (U. Link); Bayer AG, Leverkusen, Germany (G. M uller); BASF AG, Ludwigshafen, Germany
(D. Beimborn and U. Pagga); CTP, Grenoble, France (Y. Aitkin); CITI, Kurume, Japan (Y. Yakabe); OWS Ghent, Belgium (B. de Wilde); Swedish National Testing and Research Institute, Boras, Sweden (C. Lindblad); Hydrotox GmbH, Freiburg, Germany (S. Gartiser).
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