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Biodegradation and ecotoxicity evaluation of a bionolle and starch blend and its degradation products in compost
International Biodeterioration & Biodegradation 51 (2003) 77 – 81
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Biodegradation and ecotoxicity evaluation of a bionolle and starch blend and its degradation products in compost R. Jayasekaraa; ∗ , S. Sheridana , E. Lourbakosb , H. Behb , G.B.Y. Christieb , M. Jenkinsb , P.B. Halleyc , S. McGlashanc , G.T. Lonergana a Centre
for Applied Colloid and Biocolloid Science, School of Engineering and Science, Swinburne University of Technology, John Street, Hawthorn, 3122 Melbourne, Australia b CSIRO Division of Manufacturing Science and Technology, Cooperative Research Centre for International Food Manufacture and Packing Science, Clayton, Victoria 31169, Australia c Materials Characterisation and Processing Centre, The University of Queensland, Queensland 4072, Australia Received 26 November 2001; accepted 24 January 2002
Abstract A polymer based on a blend of starch and “BionolleTM ” has been prepared and tested for biodegradation in compost. The polymer was completely mineralised to carbon dioxide in 45 days. The potential toxicity of the polymer was tested against the earthworm Eisenia fetida using a modi;cation of the American Standard for Testing Materials E1976-97. The earthworms were exposed to 30 g of the polymer for 28 days and changes in weight recorded. In addition, the polymer was ;rstly degraded by the compost and the worms exposed to the breakdown products for 28 days. Di?erences in weight were also recorded. In each case the production of juveniles was noted and all earthworms were examined for pathology. The results obtained were processed statistically using a t-test. The number of juveniles, produced from the breakdown products, was highly signi;cant (P ¡ 0:001) when compared to the earthworms added to the intact polymer. There was a de;nitely signi;cant di?erence (P ¡ 0:01; t = 3:25) in change in weight between the earthworms that were exposed to the polymer directly and those that were exposed to the breakdown products. There was no indication of any pathology of any earthworms. The polymer is considered safe for this species. ? 2002 Elsevier Science Ltd. All rights reserved. Keywords: BionolleTM ; Toxicity; Eisenia fetida
1. Introduction Certain aliphatic polyesters are known to be biodegradable in the aquatic environment (Benedict et al., 1983; Nishida and Tokiwa, 1994; Yakabe and Kitano, 1994; Albertson et al., 1998; Ikada, 1999). Polyester polymers are continuously being modi;ed and a recent addition to this range is BionolleTM , which is synthesised from aliphatic dicarboxylic acids and glycols (Takiyama and Fujimaki, 1994; Yokota et al., 1994). Biodegradability of these polymers has been studied in activated sludge, soils and composts (Nishioka et al., 1994; Takiyama and Fujimaki, 1994). Despite many attractive biodegradation properties, use of BionolleTM is somewhat limited by its high cost. Accordingly, this substance has been blended with low cost starch to enhance cost competitiveness and yet maintain ∗
Corresponding author. Tel.: +61-921-48575; fax: +61-918-90834. E-mail address: [email protected] (R. Jayasekara).
other acceptable properties (Buehler et al., 1994a, b, Ratto et al., 1999; Bhattacharya, 2000). A promising formulation developed by the Cooperative Research Centre (CRC) was used in this investigation to check on the potential toxicity of this group of polymers. On mineralisation, biodegradable polymers should not produce metabolites that are toxic to fauna and Iora. One way of testing the potential toxicity of polymers during biodegradation is to use earthworms (American Society for Testing Materials E1976-97-ASTM, 1997; OECD Draft Guidelines for the testing of chemicals, 2000 (OECD, 2000)). Earthworms are considered to be important indicators of ecological health since they are a food source of higher organisms and play a role in environmental recycling by ingesting and turning over large amounts of soil and accumulating organic and inorganic compounds (Edwards and Bohlen, 1996). The toxicity of BionolleTM , and polymer formulations based on BionolleTM , has not yet been assessed. In this
0964-8305/02/$ - see front matter ? 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 4 - 8 3 0 5 ( 0 2 ) 0 0 0 9 0 - 2
R. Jayasekara et al. / International Biodeterioration & Biodegradation 51 (2003) 77 – 81
work the polymer, developed by the CRC, was tested for its toxicity to earthworms. The toxicity test, developed for detecting the accumulation of toxic soil substances in earthworms (ASTM, 1997: OECD, 2000), was impractical with “BionolleTM ” which is a polymer ;lm. Therefore, a modi;cation of the ASTM (1997) method and OECD (2000) guidelines was examined to see whether it might be capable of indicating the potential toxicity of the polymer ;lm and biodegradation products in the ;rst instance. The ASTM (1997) test recommends exposure of earthworms for 4 weeks to a soil or compost, containing various concentrations of the test substance, recovering the worms, assessing their physical condition and counting the number of progeny. In the modi;cation a single known amount of polymer ;lm was exposed to the e?ect of earthworms in a mature and highly active compost support medium. A second sample of the polymer, that had been allowed to undergo composting for 42 days prior to treatment with earthworms, was also used. This latter experiment ensured that the earthworms were also exposed to any remaining polymer and its breakdown products. The data to be examined was the change in weight, survival rate and lethal and sub-lethal pathology. By conducting the test in this way the toxic effect of the original intact polymer could be compared to any toxicity of the breakdown products. 2. Materials and methods BionolleTM Starch Blend. The starch–polyester blend was prepared as described by Buehler et al. (1994a, b). Earthworms. Ten healthy, active adult earthworms (identi;ed as healthy and fertile by a clitellum) of Eisenia fetida were used per container. Soil was removed from the exterior of the earthworms by rinsing with puri;ed water and the gut contents purged, prior to weighing, by placing the earthworms on wet ;lter paper in a glass beaker for 24 h. Excess water was then removed by absorption on ;lter paper followed by drying with tissue paper. The total weight of each group of 10 earthworms was recorded after this treatment. Treatment of materials. Six hundred grams of matured compost were placed into each of 40 glass containers of 2 dm3 capacity and distributed to a depth of 5 –6 cm. Each container had a perforated transparent cover to allow gas exchange. The compost was brought to approximately 50% moisture content by the addition of puri;ed water after the moisture content was determined using a Sartorius MA 100 moisture analyser (Jayasekara et al., 2001 or ASTM, 1997). Thirty grams of the starch blend polymer ;lm was added to 20 containers and mixed with the compost. The remaining 20 containers, without the polymer, acted as controls. The experiment followed the procedure of the ASTM test for toxicity (ASTM, 1997). Ten containers, containing the polymer ;lm (labelled T1), were provided with 10 earthworms and 10 controls (C1) lacking the polymer were also
BIODEGRADATION (%)
78
120 110 100 90 80 70 60 50 40 30 20 10 0 0
5
10
15
20 25 30 TIME (DAYS)
35
40
45
50
Fig. 1. Mineralisation of BionolleTM starch blend polymer in the multi-unit composter (Jayasekara et al., 2001) over a period of 45 days. Symbol (L) indicates polymer blend and symbol (◦) indicates cellulose.
provided with 10 earthworms. The containers were allowed to stand for 28 days. The remaining 10 containers, containing the compost and polymer (T2) along with their controls (C2), were allowed to stand for 42 days and each container was then seeded with 10 earthworms. These containers were then allowed to stand for an additional 28 days. At the end of each of the test periods all earthworms were recovered from each container. The weight of the 10 largest earthworms from each container at the end of the experimental period (following purging of gut contents) was taken and compared to the weight of the 10 original earthworms placed in the containers. Each worm was examined for biological data such as mortality, physical condition, presence of irregularities such as segmental constriction, lesions and lack of motility, using a dissecting microscope. The number of juvenile earthworms present in each container was also recorded at the end of the test period. These varied in size from those barely discernible from soil particles (1 mm in length) to others almost as large as the original adults. ◦ The external temperature was controlled at 22 C. Moisture content and the pH of a 10% suspension (1 g compost=10 mL water) were checked weekly. Compost and the polymer ;lm was mixed every 3 days by gentle inversion of containers. Biodegradation studies. The polymer ;lm was examined for biodegradation using the system described (Jayasekara et al., 2001). This apparatus measured the rate of evolution of carbon dioxide from compost treated polymeric material. Switching between compost units was e?ected by solenoid valves that directed gas Iow through a specially designed gas distribution chamber and into a carbon dioxide meter. The biodegradation of the ;lm of starch Bionolle polymer in compost was also examined optically and the extent of deterioration of the ;lm recorded photographically using a Kodak 120 digital camera.
R. Jayasekara et al. / International Biodeterioration & Biodegradation 51 (2003) 77 – 81
79
Fig. 2. Deterioration of BionolleTM starch blend polymer over a period of 35 days. There was no ;lm remaining by day 42.
3. Results and discussion Biodegradation polymer =lm. Biodegradation in the automated multi-unit composter (Jayasekara et al., 2001) as judged by complete mineralisation is shown in Fig. 1 and is compared to cellulose. It can be seen that biodegradation could readily be measured by mineralisation in the multi-unit composter. Cellulose, the standard material for comparison, was degraded by 96% (standard deviation [SD] ±1:9%) compared to the polymer blend which was fully degraded [SD ±4:1%] over
the 45 days of this run. Compared to the polymer the initial degradation of cellulose was more rapid but the complete deterioration required the 45 days. These results are con;rmed photographically for the starch polymer blend in Fig. 2, where the original intact ;lm disintegrated over the ;rst 6 weeks so that there was no visible sign of the ;lm remaining after 42 days. E>ect on weight of earthworms. Data processing involved statistics and the statistics involved comparisons of the means. The comparisons were (a) the di?erences in total weights of 10 earthworms at the start of the
80
R. Jayasekara et al. / International Biodeterioration & Biodegradation 51 (2003) 77 – 81
Table 1 Raw statistical data for weight of Eisenia fetida recorded before and after treaments
Container no.
Wt. original worms (10)
Wt. ;nal worms (10)
No. of juveniles
Control 1 C1 Compost no polymer
1 2 3 4 5 6 7 8 9 10
2.23 2.34 2.08 2.18 2.10 2.09 2.10 2.02 2.00 2.06
2.45 2.65 2.32 3.59 2.46 2.42 2.32 2.53 2.74 2.44
19 11 11 17 15 11 17 7 24 14
Control 2 C2 Compost no polymer Allowed to stand for 42 days before seeding
1 2 3 4 5 6 7 8 9 10
2.45 2.65 2.32 2.59 2.46 1.34 1.44 1.71 1.3 1.18
1.41 1.38 1.26 1.23 1.47 1.12 1.28 1.27 1.5 1.48
26 20 27 36 46 34 27 32 15 37
Container no.
Wt. original worms (10)
Wt. ;nal worms (10)
No. of juveniles
Test 1 T1 Compost plus polymer
1 2 3 4 5 6 7 8 9 10
1.93 2.09 2.19 2.31 2.20 2.24 2.08 1.88 1.74 2.20
2.17 2.33 2.57 2.60 2.62 2.39 2.68 2.25 1.96 2.40
26 46 31 29 29 21 23 23 28 30
Test 2 T2
1 2 3 4 5 6 7 8 9 10
2.42 2.23 2.53 2.74 2.54 1.51 1.19 1.25 0.93 1.24
1.38 1.85 1.47 2.1 1.85 1.82 2.24 2.52 2.17 2.28
75a 75a 75a 75a 75a 75a 75a 75a 75a 75a
Compost plus polymer Allowed to stand for 42 days before seeding
a Indicates the limit of counting of the juveniles that were obtained from the jars and were readily countable by the naked eye. Other juveniles were present but their numbers did not a?ect the statistical interpretation.
experiments and at the end of the experiments, (b) the change in weights of earthworms between the controls and the tests and (c) the di?erences in the number of progeny produced between the controls and tests. The t-test seemed to be an appropriate procedure for a statistical test for each of these areas and the null hypothesis was that there were no signi;cant di?erences between the tests and controls. The results of the trial are shown in Table 1. The ;rst set of t values shown in Table 2 involved the changes in the total weight of earthworms over 28 day ((a) above). There was an increase in the weight recorded in both the controls (C1) and in the test (T1) where the earthworms were exposed to the intact polymer. The average weight (C1—10 control earthworms) rose from 2.12 to 2:492 g and on treatment (T1) the rise was from 2.086 to 2:397 g. From a statistical viewpoint the t values exceeded the value for the signi;cance level P ¡ 0:05 but there must be some reservations concerning the comparisons because there was no method of ensuring that the original earthworms were involved in the ;nal weighing. However, as the average life cycle of the earthworms from the cocoon, through the imma◦ ture to the reproductive stage at 25 C is 51.5 days (ASTM, 1997) it seemed highly likely that the comparisons with the original earthworm were being made. The average weight of 10 earthworms on pre-degraded polymer metabolites fell from 1:944 g in the controls (C2) to 1:34 g, a result which is de;nitely signi;cant. There was a small increase in the weight in the test (T2) from 1.858 to 1:968 g, but this was not statistically signi;cant. The reservation above, that there was no method to ensure that the
original worms were ;nally weighed, is also relevant. The decrease in average weight of the controls was probably due to the disappearance of endogenous nutrients in the compost, during the 42 days of mineralisation of the polymer ;lm, prior to seeding with the earthworms. Di>erences in weight of earthworms. The t-test value for the weight di?erences over 28 days between control C1 and test T1 and between control C2 and its test T2 were not signi;cant (P ¿ 0:05 for each). This is possibly due to the limit to the potential size of growth of the earthworms prior to entering the reproductive phase. However, there were signi;cant di?erences in weight changes between the earthworms incorporated with the polymer and those added after the polymer degraded. On average, the earthworms in the latter part of the experiment were smaller. This may reasonably be attributed to the mineralisation of the compost ingredients during the pre-treatment of the polymer. Thus, it seems likely that the polymer degrades to readily assimilated metabolites prior to the exposure to the earthworms, and potential nutrients in the compost are consumed during this stage. E>ect on earthworm Number. Despite a 51.5 day reproductive cycle (ASTM,1997) it was found that juveniles were present in the compost environment (Table 1). The di?erences in numbers between the untreated polymer and its control, the predigested polymer and its control and the di?erences between the test and the controls for each treatment (after 28 days) was highly signi;cant (P ¡ 0:001). This means that pre-digestion of the polymer shows a highly signi;cant di?erence over undigested material. The null
R. Jayasekara et al. / International Biodeterioration & Biodegradation 51 (2003) 77 – 81
81
Table 2 t Scores for comparisons of the various categories in the experiments
Type
Comparisonsa
T valueb
Interpretationc
1 2 3 4
Total weights
C10 T10 C20 T20
6.88 3.37 3.1 0.45
Highly signi;cant De;nitely signi;cant De;nitely signi;cant Not signi;cant
5 6 7
Weight di?erences
1 above vs. 2 above 3 above vs. 4 above (1–2) above vs. (3– 4) above
0.12 1.97 3.25
Not signi;cant Not signi;cant De;nitely signi;cant
Di?erences in numbers
C1 vs. T1 C2 vs. T2 (T1–C1) vs. (T2–C2)
5.19 15.9 7.72
Highly signi;cant Highly signi;cant Highly signi;cant
Experiment No.
8 9 10
vs. C128 vs. T228 vs. C228 vs. T228
a Subscripts
indicate the number of days of the experiment. the comparison of means of two samples following t values apply t = 2:09 at the 5% probability level (P ¡ 0:05): t = 2:53 at the 2% probability level (P; 0:02): t = 2:85 at the 1% probability level (P; 0:01): t = 3:55 at the 0.2% probability level (P ¡ 0:002) and t = 3:85 at the 0.1% probability level (P, 0.001). c Interpretations: If t is greater than P ¡ 0:05 the result is probably signi;cant. If t is greater than P ¡ 0:01 the result is de;nitely signi;cant and if t is greater P ¡ 0:001 the result is highly signi;cant. This means that the results obtained will occur by chance once in every 20, 100 and 1000, respectively, times if the experiment is repeated. Source: Lindley and Miller (1962) Cambridge University Statistical Tables. b For
hypothesis must be rejected in these instances. The hypothesis arising from this statistical treatment is that the pre-treatment of the starch polymer ;lm by the compost leads to production of metabolites that are highly bene;cial to the reproduction of the earthworm. Pathology. All earthworms were examined for mortality and no earthworm showed any evidence of lesions or distress and at the end of the test they were all alive and well and motile. From these results it must be concluded that this polymer breaks down to some compounds which are non-toxic to the worms and actually aid their reproductive capacity in compost. It is concluded that this polymer is environmentally safe based on the criterion of number of juveniles and the duration of the test. References Albertson, A.-C., Renstad, R., Erlandasson, B., Eldsater, C., Karklsson, S., 1998. E?ect of processing additives on (bio)degradability of ;lm-blown poly(-caprolactone). Journal of Applied Polymer Science 70, 61–74. American Society for Testing Materials E1676-97, 1997. Standard guide for conducting laboratory soil toxicity or bio-accumulation tests with the earthworm. Eisinia fetida 1997. Benedict, C.V., Cameron, J.A., Huang, S.J., 1983. Polycaprolactone degradation by mixed and pure cultures of bacteria and yeast. Journal of Applied Polymer Science 28, 335–342. Bhattacharya, A., 2000. Radiation and industrial polymers. Progress in Polymer Science 25, 371–401. Buehler, F.S., Schmid, E., Schultz, H.-J., 1994a. Starch=polymer mixture, process for the preparation thereof, and products obtainable therefrom. US Patent 5, 346, 936.
Buehler, S., Cassanova, J., Hansjorg, E., Schultz, H.-J., 1994b. Process and apparatus for producing starch melt and products obtainable by this process. US Patent 5, 316, 578. Edwards, C.A., Bohlen, P.J., 1996. The Biology and Ecology of Earthworms, 3rd Edition. Chapman & Hall, London, Glasgow, Weinheim, New York, Tokyo, Melbourne, Madrid, pp. 292–298. Ikada, E., 1999. Electron microscope observation of the biodegradation of polymers. Journal of Environmental Polymer Degradation 7, 197–201. Jayasekara, R., Lonergan, G.T., Harding, I., Bowater, I., Halley, P., Christie, G.B., 2001. An automated multi-unit composting facility for biodegradability evaluations. Journal of Chemical Technology and Biotechnology 76, 411–417. Lindley, D.V., Miller, J.C.P., 1962. Cambridge University Statistical Tables. Cambridge University Press, Cambridge. Nishida, H., Tokiwa, Y., 1994. Con;rmation of anaerobic poly(2-oxepanone) degrading micro-organisms in environments. Chemical Letters 1293–1296. Nishioka, M., Tuzuki, T., Wanajyo, Y., Oonami, H., Horiuchi, T., 1994. Biodegradation of BIONOLLE. In: Doi, Y., Fukuda, K (Eds), Biodegradable plastics and polymers. Elsevier Science, Amsterdam, pp. 584 –590. OECD Guidelines for the testing of chemicals proposal for a new guideline, 2000. Earthworm reproduction test Eisinia fertida =anmdrei. Draft document, January 2000. Ratto, J., Stenhouose, P.J., Auerbach, M., Mitchell, J., Farrell, R., 1999. Processing performance and biodegradability of a thermoplastic aliphatic polyester=starch system. Polymer 40, 6777–6788. Takiyama, E., Fujimaki, 1994. Bionolle biodegradable plastic through chemical synthesis. In: Doi, Y., Fukuda, K (Eds.), Biodegradable Plastics and Polymers. Elsevier Science, Amsterdam, pp. 150–174. Yakabe, Y., Kitano, M., 1994. Evaluation of biodegradability of biodegradable plastics in activated sludge. In: Doi, Y., Fukuda, K (Eds.), Biodegradable Plastics and Polymers. Elsevier Science, Amsterdam, pp. 331–336. Yokota, Y. Ishioka, R., Moteki, Y., Watanabe, N., 1994. Physical properies and processsabilities of biodergradable plastics “BIONOLLE”.
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Biodegradation and ecotoxicity evaluation of a bionolle and starch blend and its degradation products in compost R. Jayasekaraa; ∗ , S. Sheridana , E. Lourbakosb , H. Behb , G.B.Y. Christieb , M. Jenkinsb , P.B. Halleyc , S. McGlashanc , G.T. Lonergana a Centre
for Applied Colloid and Biocolloid Science, School of Engineering and Science, Swinburne University of Technology, John Street, Hawthorn, 3122 Melbourne, Australia b CSIRO Division of Manufacturing Science and Technology, Cooperative Research Centre for International Food Manufacture and Packing Science, Clayton, Victoria 31169, Australia c Materials Characterisation and Processing Centre, The University of Queensland, Queensland 4072, Australia Received 26 November 2001; accepted 24 January 2002
Abstract A polymer based on a blend of starch and “BionolleTM ” has been prepared and tested for biodegradation in compost. The polymer was completely mineralised to carbon dioxide in 45 days. The potential toxicity of the polymer was tested against the earthworm Eisenia fetida using a modi;cation of the American Standard for Testing Materials E1976-97. The earthworms were exposed to 30 g of the polymer for 28 days and changes in weight recorded. In addition, the polymer was ;rstly degraded by the compost and the worms exposed to the breakdown products for 28 days. Di?erences in weight were also recorded. In each case the production of juveniles was noted and all earthworms were examined for pathology. The results obtained were processed statistically using a t-test. The number of juveniles, produced from the breakdown products, was highly signi;cant (P ¡ 0:001) when compared to the earthworms added to the intact polymer. There was a de;nitely signi;cant di?erence (P ¡ 0:01; t = 3:25) in change in weight between the earthworms that were exposed to the polymer directly and those that were exposed to the breakdown products. There was no indication of any pathology of any earthworms. The polymer is considered safe for this species. ? 2002 Elsevier Science Ltd. All rights reserved. Keywords: BionolleTM ; Toxicity; Eisenia fetida
1. Introduction Certain aliphatic polyesters are known to be biodegradable in the aquatic environment (Benedict et al., 1983; Nishida and Tokiwa, 1994; Yakabe and Kitano, 1994; Albertson et al., 1998; Ikada, 1999). Polyester polymers are continuously being modi;ed and a recent addition to this range is BionolleTM , which is synthesised from aliphatic dicarboxylic acids and glycols (Takiyama and Fujimaki, 1994; Yokota et al., 1994). Biodegradability of these polymers has been studied in activated sludge, soils and composts (Nishioka et al., 1994; Takiyama and Fujimaki, 1994). Despite many attractive biodegradation properties, use of BionolleTM is somewhat limited by its high cost. Accordingly, this substance has been blended with low cost starch to enhance cost competitiveness and yet maintain ∗
Corresponding author. Tel.: +61-921-48575; fax: +61-918-90834. E-mail address: [email protected] (R. Jayasekara).
other acceptable properties (Buehler et al., 1994a, b, Ratto et al., 1999; Bhattacharya, 2000). A promising formulation developed by the Cooperative Research Centre (CRC) was used in this investigation to check on the potential toxicity of this group of polymers. On mineralisation, biodegradable polymers should not produce metabolites that are toxic to fauna and Iora. One way of testing the potential toxicity of polymers during biodegradation is to use earthworms (American Society for Testing Materials E1976-97-ASTM, 1997; OECD Draft Guidelines for the testing of chemicals, 2000 (OECD, 2000)). Earthworms are considered to be important indicators of ecological health since they are a food source of higher organisms and play a role in environmental recycling by ingesting and turning over large amounts of soil and accumulating organic and inorganic compounds (Edwards and Bohlen, 1996). The toxicity of BionolleTM , and polymer formulations based on BionolleTM , has not yet been assessed. In this
0964-8305/02/$ - see front matter ? 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 4 - 8 3 0 5 ( 0 2 ) 0 0 0 9 0 - 2
R. Jayasekara et al. / International Biodeterioration & Biodegradation 51 (2003) 77 – 81
work the polymer, developed by the CRC, was tested for its toxicity to earthworms. The toxicity test, developed for detecting the accumulation of toxic soil substances in earthworms (ASTM, 1997: OECD, 2000), was impractical with “BionolleTM ” which is a polymer ;lm. Therefore, a modi;cation of the ASTM (1997) method and OECD (2000) guidelines was examined to see whether it might be capable of indicating the potential toxicity of the polymer ;lm and biodegradation products in the ;rst instance. The ASTM (1997) test recommends exposure of earthworms for 4 weeks to a soil or compost, containing various concentrations of the test substance, recovering the worms, assessing their physical condition and counting the number of progeny. In the modi;cation a single known amount of polymer ;lm was exposed to the e?ect of earthworms in a mature and highly active compost support medium. A second sample of the polymer, that had been allowed to undergo composting for 42 days prior to treatment with earthworms, was also used. This latter experiment ensured that the earthworms were also exposed to any remaining polymer and its breakdown products. The data to be examined was the change in weight, survival rate and lethal and sub-lethal pathology. By conducting the test in this way the toxic effect of the original intact polymer could be compared to any toxicity of the breakdown products. 2. Materials and methods BionolleTM Starch Blend. The starch–polyester blend was prepared as described by Buehler et al. (1994a, b). Earthworms. Ten healthy, active adult earthworms (identi;ed as healthy and fertile by a clitellum) of Eisenia fetida were used per container. Soil was removed from the exterior of the earthworms by rinsing with puri;ed water and the gut contents purged, prior to weighing, by placing the earthworms on wet ;lter paper in a glass beaker for 24 h. Excess water was then removed by absorption on ;lter paper followed by drying with tissue paper. The total weight of each group of 10 earthworms was recorded after this treatment. Treatment of materials. Six hundred grams of matured compost were placed into each of 40 glass containers of 2 dm3 capacity and distributed to a depth of 5 –6 cm. Each container had a perforated transparent cover to allow gas exchange. The compost was brought to approximately 50% moisture content by the addition of puri;ed water after the moisture content was determined using a Sartorius MA 100 moisture analyser (Jayasekara et al., 2001 or ASTM, 1997). Thirty grams of the starch blend polymer ;lm was added to 20 containers and mixed with the compost. The remaining 20 containers, without the polymer, acted as controls. The experiment followed the procedure of the ASTM test for toxicity (ASTM, 1997). Ten containers, containing the polymer ;lm (labelled T1), were provided with 10 earthworms and 10 controls (C1) lacking the polymer were also
BIODEGRADATION (%)
78
120 110 100 90 80 70 60 50 40 30 20 10 0 0
5
10
15
20 25 30 TIME (DAYS)
35
40
45
50
Fig. 1. Mineralisation of BionolleTM starch blend polymer in the multi-unit composter (Jayasekara et al., 2001) over a period of 45 days. Symbol (L) indicates polymer blend and symbol (◦) indicates cellulose.
provided with 10 earthworms. The containers were allowed to stand for 28 days. The remaining 10 containers, containing the compost and polymer (T2) along with their controls (C2), were allowed to stand for 42 days and each container was then seeded with 10 earthworms. These containers were then allowed to stand for an additional 28 days. At the end of each of the test periods all earthworms were recovered from each container. The weight of the 10 largest earthworms from each container at the end of the experimental period (following purging of gut contents) was taken and compared to the weight of the 10 original earthworms placed in the containers. Each worm was examined for biological data such as mortality, physical condition, presence of irregularities such as segmental constriction, lesions and lack of motility, using a dissecting microscope. The number of juvenile earthworms present in each container was also recorded at the end of the test period. These varied in size from those barely discernible from soil particles (1 mm in length) to others almost as large as the original adults. ◦ The external temperature was controlled at 22 C. Moisture content and the pH of a 10% suspension (1 g compost=10 mL water) were checked weekly. Compost and the polymer ;lm was mixed every 3 days by gentle inversion of containers. Biodegradation studies. The polymer ;lm was examined for biodegradation using the system described (Jayasekara et al., 2001). This apparatus measured the rate of evolution of carbon dioxide from compost treated polymeric material. Switching between compost units was e?ected by solenoid valves that directed gas Iow through a specially designed gas distribution chamber and into a carbon dioxide meter. The biodegradation of the ;lm of starch Bionolle polymer in compost was also examined optically and the extent of deterioration of the ;lm recorded photographically using a Kodak 120 digital camera.
R. Jayasekara et al. / International Biodeterioration & Biodegradation 51 (2003) 77 – 81
79
Fig. 2. Deterioration of BionolleTM starch blend polymer over a period of 35 days. There was no ;lm remaining by day 42.
3. Results and discussion Biodegradation polymer =lm. Biodegradation in the automated multi-unit composter (Jayasekara et al., 2001) as judged by complete mineralisation is shown in Fig. 1 and is compared to cellulose. It can be seen that biodegradation could readily be measured by mineralisation in the multi-unit composter. Cellulose, the standard material for comparison, was degraded by 96% (standard deviation [SD] ±1:9%) compared to the polymer blend which was fully degraded [SD ±4:1%] over
the 45 days of this run. Compared to the polymer the initial degradation of cellulose was more rapid but the complete deterioration required the 45 days. These results are con;rmed photographically for the starch polymer blend in Fig. 2, where the original intact ;lm disintegrated over the ;rst 6 weeks so that there was no visible sign of the ;lm remaining after 42 days. E>ect on weight of earthworms. Data processing involved statistics and the statistics involved comparisons of the means. The comparisons were (a) the di?erences in total weights of 10 earthworms at the start of the
80
R. Jayasekara et al. / International Biodeterioration & Biodegradation 51 (2003) 77 – 81
Table 1 Raw statistical data for weight of Eisenia fetida recorded before and after treaments
Container no.
Wt. original worms (10)
Wt. ;nal worms (10)
No. of juveniles
Control 1 C1 Compost no polymer
1 2 3 4 5 6 7 8 9 10
2.23 2.34 2.08 2.18 2.10 2.09 2.10 2.02 2.00 2.06
2.45 2.65 2.32 3.59 2.46 2.42 2.32 2.53 2.74 2.44
19 11 11 17 15 11 17 7 24 14
Control 2 C2 Compost no polymer Allowed to stand for 42 days before seeding
1 2 3 4 5 6 7 8 9 10
2.45 2.65 2.32 2.59 2.46 1.34 1.44 1.71 1.3 1.18
1.41 1.38 1.26 1.23 1.47 1.12 1.28 1.27 1.5 1.48
26 20 27 36 46 34 27 32 15 37
Container no.
Wt. original worms (10)
Wt. ;nal worms (10)
No. of juveniles
Test 1 T1 Compost plus polymer
1 2 3 4 5 6 7 8 9 10
1.93 2.09 2.19 2.31 2.20 2.24 2.08 1.88 1.74 2.20
2.17 2.33 2.57 2.60 2.62 2.39 2.68 2.25 1.96 2.40
26 46 31 29 29 21 23 23 28 30
Test 2 T2
1 2 3 4 5 6 7 8 9 10
2.42 2.23 2.53 2.74 2.54 1.51 1.19 1.25 0.93 1.24
1.38 1.85 1.47 2.1 1.85 1.82 2.24 2.52 2.17 2.28
75a 75a 75a 75a 75a 75a 75a 75a 75a 75a
Compost plus polymer Allowed to stand for 42 days before seeding
a Indicates the limit of counting of the juveniles that were obtained from the jars and were readily countable by the naked eye. Other juveniles were present but their numbers did not a?ect the statistical interpretation.
experiments and at the end of the experiments, (b) the change in weights of earthworms between the controls and the tests and (c) the di?erences in the number of progeny produced between the controls and tests. The t-test seemed to be an appropriate procedure for a statistical test for each of these areas and the null hypothesis was that there were no signi;cant di?erences between the tests and controls. The results of the trial are shown in Table 1. The ;rst set of t values shown in Table 2 involved the changes in the total weight of earthworms over 28 day ((a) above). There was an increase in the weight recorded in both the controls (C1) and in the test (T1) where the earthworms were exposed to the intact polymer. The average weight (C1—10 control earthworms) rose from 2.12 to 2:492 g and on treatment (T1) the rise was from 2.086 to 2:397 g. From a statistical viewpoint the t values exceeded the value for the signi;cance level P ¡ 0:05 but there must be some reservations concerning the comparisons because there was no method of ensuring that the original earthworms were involved in the ;nal weighing. However, as the average life cycle of the earthworms from the cocoon, through the imma◦ ture to the reproductive stage at 25 C is 51.5 days (ASTM, 1997) it seemed highly likely that the comparisons with the original earthworm were being made. The average weight of 10 earthworms on pre-degraded polymer metabolites fell from 1:944 g in the controls (C2) to 1:34 g, a result which is de;nitely signi;cant. There was a small increase in the weight in the test (T2) from 1.858 to 1:968 g, but this was not statistically signi;cant. The reservation above, that there was no method to ensure that the
original worms were ;nally weighed, is also relevant. The decrease in average weight of the controls was probably due to the disappearance of endogenous nutrients in the compost, during the 42 days of mineralisation of the polymer ;lm, prior to seeding with the earthworms. Di>erences in weight of earthworms. The t-test value for the weight di?erences over 28 days between control C1 and test T1 and between control C2 and its test T2 were not signi;cant (P ¿ 0:05 for each). This is possibly due to the limit to the potential size of growth of the earthworms prior to entering the reproductive phase. However, there were signi;cant di?erences in weight changes between the earthworms incorporated with the polymer and those added after the polymer degraded. On average, the earthworms in the latter part of the experiment were smaller. This may reasonably be attributed to the mineralisation of the compost ingredients during the pre-treatment of the polymer. Thus, it seems likely that the polymer degrades to readily assimilated metabolites prior to the exposure to the earthworms, and potential nutrients in the compost are consumed during this stage. E>ect on earthworm Number. Despite a 51.5 day reproductive cycle (ASTM,1997) it was found that juveniles were present in the compost environment (Table 1). The di?erences in numbers between the untreated polymer and its control, the predigested polymer and its control and the di?erences between the test and the controls for each treatment (after 28 days) was highly signi;cant (P ¡ 0:001). This means that pre-digestion of the polymer shows a highly signi;cant di?erence over undigested material. The null
R. Jayasekara et al. / International Biodeterioration & Biodegradation 51 (2003) 77 – 81
81
Table 2 t Scores for comparisons of the various categories in the experiments
Type
Comparisonsa
T valueb
Interpretationc
1 2 3 4
Total weights
C10 T10 C20 T20
6.88 3.37 3.1 0.45
Highly signi;cant De;nitely signi;cant De;nitely signi;cant Not signi;cant
5 6 7
Weight di?erences
1 above vs. 2 above 3 above vs. 4 above (1–2) above vs. (3– 4) above
0.12 1.97 3.25
Not signi;cant Not signi;cant De;nitely signi;cant
Di?erences in numbers
C1 vs. T1 C2 vs. T2 (T1–C1) vs. (T2–C2)
5.19 15.9 7.72
Highly signi;cant Highly signi;cant Highly signi;cant
Experiment No.
8 9 10
vs. C128 vs. T228 vs. C228 vs. T228
a Subscripts
indicate the number of days of the experiment. the comparison of means of two samples following t values apply t = 2:09 at the 5% probability level (P ¡ 0:05): t = 2:53 at the 2% probability level (P; 0:02): t = 2:85 at the 1% probability level (P; 0:01): t = 3:55 at the 0.2% probability level (P ¡ 0:002) and t = 3:85 at the 0.1% probability level (P, 0.001). c Interpretations: If t is greater than P ¡ 0:05 the result is probably signi;cant. If t is greater than P ¡ 0:01 the result is de;nitely signi;cant and if t is greater P ¡ 0:001 the result is highly signi;cant. This means that the results obtained will occur by chance once in every 20, 100 and 1000, respectively, times if the experiment is repeated. Source: Lindley and Miller (1962) Cambridge University Statistical Tables. b For
hypothesis must be rejected in these instances. The hypothesis arising from this statistical treatment is that the pre-treatment of the starch polymer ;lm by the compost leads to production of metabolites that are highly bene;cial to the reproduction of the earthworm. Pathology. All earthworms were examined for mortality and no earthworm showed any evidence of lesions or distress and at the end of the test they were all alive and well and motile. From these results it must be concluded that this polymer breaks down to some compounds which are non-toxic to the worms and actually aid their reproductive capacity in compost. It is concluded that this polymer is environmentally safe based on the criterion of number of juveniles and the duration of the test. References Albertson, A.-C., Renstad, R., Erlandasson, B., Eldsater, C., Karklsson, S., 1998. E?ect of processing additives on (bio)degradability of ;lm-blown poly(-caprolactone). Journal of Applied Polymer Science 70, 61–74. American Society for Testing Materials E1676-97, 1997. Standard guide for conducting laboratory soil toxicity or bio-accumulation tests with the earthworm. Eisinia fetida 1997. Benedict, C.V., Cameron, J.A., Huang, S.J., 1983. Polycaprolactone degradation by mixed and pure cultures of bacteria and yeast. Journal of Applied Polymer Science 28, 335–342. Bhattacharya, A., 2000. Radiation and industrial polymers. Progress in Polymer Science 25, 371–401. Buehler, F.S., Schmid, E., Schultz, H.-J., 1994a. Starch=polymer mixture, process for the preparation thereof, and products obtainable therefrom. US Patent 5, 346, 936.
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