Biodegradation Process of Polymers
4.3 Recommendations and Standard Procedures for Biotests
4.3.1 Bioassays with Higher Plants
Since plants are in most cases the primary target organisms for agricultural applications the methods and infl uencing parameters of plant biotests will be described in more detail than for all the other test organisms.
4.3.1.1 Test Set Up and General Conditions
The OECD method 208 [32] requires the application of at least three tests in parallel using three different plant species. That seems to be justifi ed, since the grouped species are sensitive to different inhibition mechanisms. Such tests could be varied for the determination of the germination rate or of the plant growth (biomass production) or for both effects at the same time.
For the determination of the germination rate, a known number of seeds is put on top of the sample and watered properly. After the species specifi c germination time the number of young plants is counted and compared with the germination rate in the reference
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Table 4.2 Overview about the most suitable bioassays for ecotoxicity testing of polymers during and after their biodegradation
Test organism Sample types Standard/literature Comments Higher plants, terrestrial
(cress, millet, rape etc.)
compost and soil
OECD 208 [32];
ISO 11269-1 and ISO 11269-2 [33]
many species available Higher plants, aquatic
(Lemna sp.)
freshwater and sediments
OECD-draft [34] currently not standardised Fish (various species) fresh- and
seawater
OECD 203 [35], 204 [36], 210 [37];
ISO 7346 [38];
DIN 38412 L15 [39]
static or fl ow through design
Earthworm (Eisenia foetida) soil and sediments
OECD 207 [40];
ISO 11268 [41]
not suitable in presence of digestible materials Collembola (Folsomia
candita)
compost and soil
ISO 11267 [42]
Protozoa (ciliates) soil Berthold [43] not standardised Protozoa (Colpoda mauposi) freshwater DEV L10 [44] designed for
wastewater Crustaceae (Daphnia
magna)
freshwater OECD 202 [45], 211 [46]; ISO 6341 [47], 10706 [48]; DIN EN ISO 5667-16 [49], 38412 L30 [50]
acute and chronic
Crustaceae (Artemia sp.) seawater ISO 14669 [51]
Algae (Scenedesmus subsp., Selensatrum cap., Chlorella sp.)
freshwater OECD 201 [52];
ISO 8692 [53];
DIN 38412 L33 [54]
test used very often
Algae (Skeletonema costatum, Phaeodactylum tricornutum)
seawater ISO 10253 [55];
DIN 38412 L45 [56]
Bacteria (Pseudomonas putida)
freshwater ISO 10712 [57]
Luminescent bacteria (Vibrio fi scheri, Photobacterium sp.)
sea-water ISO 11348 [58] very short exposure time
Enzymic activity soil and sediment
OECD 216 [59], 217 [60]; ISO 9509 [61]
measurement of N- and C-transformation Various organisms and DNA - OECD 471 - 486 [62] several mutagenity
tests
Multispecies tests all environments Calow [24] not standardised
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117 substrate. The test time could be extended for two or three weeks (depending on the plant species) and the grown plants could then be harvested for determination of the biomass production. Similarly to the germination rate, the biomasses obtained from the samples are also compared with those from the reference substrate.
For the determination of the plant growth rate, young plants are pre-cultivated in a reference substrate and are then transferred to the prepared samples. That procedure requires some experience since the small roots must not be injured and the amounts of adhering reference substrate should be as small as possible. After a typical growth time the plants are harvested and the biomass produced is evaluated.
In all variants the samples (150 g to 250 g) are placed into trays made of polyethylene or glass. A thin layer of washed sand on the bottom can form a drain layer and a very thin layer on the top (spread carefully over the seed) avoids the drifting of the seed during the watering.
The reference matrix for plant tests should be chosen with care. When investigating the ecotoxic effects of biodegradable materials these are commonly mixed with compost or soil for the biodegradation test. The original matrix should be used as reference and for dilution of the samples. It is unavoidable that the test plants will grow differently in each compost batch and each type of soil. Standardised matrices, such as are given in the standard methods, should be avoided since those are optimised for toxicity tests of chemicals, added in defi ned amounts and not treated any further before the start of the biotests. For the calculation of ecotoxic effects deriving from the degradation process of polymer materials the difference to results from the same matrix without any additions is needed.
4.3.1.2 Special Test Conditions
The watering of the tests has a major impact on the plant growth and should be done carefully. Over the whole period of the plant growth the water content should be kept as constant as possible and at an optimised level. If the water content is too low, the ion transport from the roots to the leaves is inhibited or the plants will die from thirst. If the water content is too high, all the pores in the sample will be fl ooded and the oxygen transfer (normally by diffusion) will be interrupted. Anoxic or even anaerobic conditions will be the consequence. Again the plant growth will be inhibited or they will die. The water content should therefore be kept between 70% and 100% of the water holding capacity of the sample matrix. This fact is not mentioned in the standard methods. The infl uence of the water content on the plant growth in bioassays is shown in Figure 4.3. To keep the water content in the recommended range it is helpful to know the weight of each test tray calculated at 70% and 100% water saturation. During the test time whenever the lower weight is reached, water is added up to the weight of 100% saturation.
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Current research results have shown that the response of the three species cress, millet and rape may be spread over a wide range using soil samples, which have degraded different biogenic and synthetic polymers. In our group we found that typical physical and chemical parameters determined from such soil samples did not correlate strictly with the observed inhibition of the plant growth. Some of the substances responsible for the effects may not be detected by a conventional soil analysis and remain unknown.
Following Chen and Inbar [63] and interpreting results obtained by our group [64] the most probable causes may be:
• decrease of the oxygen content in the soil pores by microbial consumption due to the degradation process and in the consequence a decrease of the redox-potential
• formation of fatty acids, especially formic, acetic and propionic acid as typical metabolic intermediates
• decrease of the pH value for the same reason as above
• formation of stable toxic metabolites and degradation residues which are in most cases very diffi cult to detect
• a shift in the composition of the microbial community and an enrichment of potentially plant pathogenic organisms (or organisms with allelopathic properties).
Figure 4.3 Infl uence of the amount of water used for the periodical watering on the plant growth in bioassays with standardised substrate alone and with 2% starch
added. Derived from [64]
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119 The fi rst three causes for an inhibition of plant growth are temporal effects, directly connected to increased microbial activity during the degradation process and may end at the time the degradation is completed. The last two causes could be of temporal nature but could be present over a longer time.