Polymer Degradation and Stability xxx (2014) 1e7

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Polymer Degradation and Stability

journal homepage: www.elsevier.com/locate/polydegstab

An in vitro crop plant ecotoxicity test for agricultural bioplastic

constituents
L. Martin-Closas*, R. Botet, A.M. Pelacho
Dpt. Horticulture, Botany and Gardening, University of Lleida, Avda. Alcalde Rovira Roure 191, 25198 Lleida, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Plastic mulches are widely used in agriculture to improve production, mainly in vegetable crops. Their
Received 7 December 2013 main drawback is the generation of residues that are hard to manage. Therefore the substitution of
Received in revised form traditional plastics by renewable and biodegradable polymers is an environmentally friendly improve-
12 March 2014
ment. However, compounds released during (bio)degradation of the mulches may remain in the soil.
Accepted 26 March 2014
Consequently, standard ecotoxicity tests are required to ensure the biomaterials’ ecosafety. Unfortu-
Available online xxx
nately, ecotoxicity tests for terrestrial plants, and specifically for the plant species frequently cultivated
with mulches, are poorly developed. Furthermore, most of these tests report seedling emergence and
Keywords:
Adipic acid
early plant growth, but germination and plant growth have different requirements, and plant growth
Succinic acid inhibition by compounds not affecting germination has been frequently reported. Other limitations of
Lactic acid;1,4-butanediol ecotoxicity tests are related to environmental variability, interactions of soils/substrates with the
Lettuce chemicals, and to the limited monitoring of plant development over time, especially for roots.
Tomato The aim of this work has been to develop an in vitro controlled system for testing the ecotoxicity of
plastic constituents putatively delivered to the soil during mulch biodegradation on crop plants.
Germination and growth of lettuce and tomato were monitored over time in response to adipic, succinic
and lactic acids, and to 1,4-butanediol, in concentrations 5e500 mg l
1. Although germination was not
influenced by most treatments, significant effects were manifested later in plant development. The
sensitivity of the system was higher than in standard short-term assays. Results in lettuce and tomato
were not substantially different, but indicative of the need to test the precise species targeted. Overall,
adipic acid inhibited growth, succinic acid had no effect, and butanediol enhanced growth to some
extent. Lactic acid requires further investigation. We highlight the convenience of the system for
monitoring root development; roots were more sensitive to the chemicals than shoots and leaves. Proline
is shown as a potential marker for ecotoxicoxicity. The in vitro system is proposed as a simple and
reliable method to test for ecotoxicity in terrestrial plants.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction replacement of traditional plastics, mostly polyethylene mulches,

Plastics are widely used in agriculture but produce residues. The
management of the agricultural packaging and structural elements’
residues, though costly, can be conveniently accomplished. How-
ever, plastics used in the fields, with mulches as the most relevant
ones, are exposed to climatic agents, break into pieces and mix with
the soil. More often than not, these agricultural plastic bits may
remain buried for decades in the field: they may be swept away to
enter into surrounding natural ecosystems and eventually reach
the marine environment. With this far-reaching consequences, the
* Corresponding author. Tel.: þ34 973702567.
E-mail address: martin@hbj.udl.cat (L. Martin-Closas).
with renewable and biodegradable polymers has been particularly
welcome. Several approved brands of totally or partially biode-
gradable mulches have already proven reliable in terms of agri-
cultural performance [1]. In the development of these new
biodegradable plastics for agriculture, the main focus has been on
the potential biodegradation of the plastic.
A further required step is to determine the consequences of the
biodegradation processes in the field environment, where the
material will repeatedly break down and deliver their many and
diverse constituents to the soil. It is important to emphasize that
the molecules released from plastics may frequently play a role in
plant metabolism, or are allelochemicals themselves that regulate
interactions between plants [2]. Equally, it is not unlikely that some
sub-products of the mulches’ biodegradation process may be

http://dx.doi.org/10.1016/j.polymdegradstab.2014.03.037
0141-3910/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Martin-Closas L, et al., An in vitro crop plant ecotoxicity test for agricultural bioplastic constituents, Polymer
Degradation and Stability (2014), http://dx.doi.org/10.1016/j.polymdegradstab.2014.03.037
2 L. Martin-Closas et al. / Polymer Degradation and Stability xxx (2014) 1e7
absorbed by the next generation of crop plants, where they may
have ecotoxic effects and may decrease agricultural productivity in
the long term.
Different methodologies for studying ecotoxicity in biodegra-
dation processes have been developed. Usually, a soil environment
related organism (earthworms, insects, algae, bacteria, protozoa
or plants) is exposed to media where the plastics have been bio-
degraded, and growth or reproductive rate is monitored. The se-
lection of the target organism is essential. Although at field-level
the organisms that live in the soil environment are important to
identify toxicity, the primary focus of agriculture is the crop itself
[3]. In the case of biodegradable mulching, target species should
be chosen among those in which these materials are usually
applied.
The biodegradation process, and thus the deliverey of putative
toxic compounds to the soil, is highly dependent on soil properties
and environmental conditions under which it takes place. There-
fore, it is quite difficult to select a broad-range soil medium for the
agricultural mulches’ ecotoxicity bioassays. Furthermore, the
polymer intermediates released along the biodegradations process
of the mulches may be ecotoxic or not, depending on the stage of
degradation of the material and on the developmental phase of the
plant. As a result, ecotoxicity studies are considerably complex.
Among the tests, the OECD 208 Guideline for testing of chemicals
(Table 1) has been developed for assessing the toxicity of chemicals
applied to plants by mixing them with the soil or by spraying the
chemical onto the soil surface. This guideline provides the main
outlines and the general criteria to build up ecotoxicological tests,
and most of the current tests (Table 1) are to be taken as an
approach, all having limitations and restrictions.
In order to better address the current constraints, new meth-
odologies for the assessment of potential ecotoxic chemicals are
necessary. Ecotoxicity can be determined by exposure of the plants
to the constituents of the materials, or to the intermediate products
resulting from the biodegradation process. Care should be taken to
avoid variations inherent to the soil interface by using a stan-
dardized test medium. Furthermore, the methodology should allow
for simulating a broad range of environmental conditions and also
for monitoring germination, growth and root development over
time.
This need has inspired us to investigate a simple and controlled
in vitro system to demonstrate the potential effects on plant growth
and development that may result from the accumulation of some of
these compounds in the field. The aim of the present work is to
develop an in vitro method to test the potential ecotoxic effects of
selected plastic constituents that can be released as a result of a
biodegradation process on two widely-cultivated plants. This is a
first step in evaluating the complex and long-term effects of
biodegradable products used in agriculture.
Table 1
Main tests using plants for the assessment of ecotoxic effects of chemicals in the soil.
Methodology
ISO 11269-1:1993 Soil quality. Determination of the effects of
pollutants on soil flora. Part 1. Method for
the measurement of inhibition of root growth
ISO 11269-2:1995 Soil quality. Determination of the effects of
pollutants on soil flora. Part 2. Effect of chemicals
on the emergence and growth of higher plants
ISO 22030:2005 Soil quality. Biological methods. Chronic toxicity
in higher plants
ISO 17126:2005 Soil quality. Determination of the effects of pollutants
on soil flora. Screening test for emergence of lettuce
seedlings (Lactuca sativa L.)
OECD 208 Guideline for the testing of chemicals
Please cite this article in press as: Martin-Closas L, et al., An in vitro crop plant ecotoxicity test for agricultural bioplastic constituents, Polymer
Degradation and Stability (2014), http://dx.doi.org/10.1016/j.polymdegradstab.2014.03.037
2. Materials and methods
2.1. Tissue culture media and treatments preparation
A Murashige and Skoog (MS) [4] broad range culture medium
with 3% sucrose was prepared with pH adjusted to 5.7. After
autoclaving for 20 min at 121  C, when media temperature was
50  C, solutions of chemicals frequently present in bioplastics were
filter sterilized and added to final concentrations of 0, 5, 50 and
500 mg l
1 in the culture media. Media were distributed into sterile
culture tubes. The chemicals tested were adipic acid (Acros Or-
ganics, 99%), succinic acid (Acros Organics, 99%), 1,4-butanediol
(Acros Organics, 99%) and L(þ)-lactic acid (Acros Organics, 90%
water solution).
In a second separate experiment specifically for lettuce, an
additional set of adipic and succinic acid treatments were prepared.
Half of the treatments were made as above, while for the other half
the pH was adjusted pH to 5.1 after autoclaving, so this pH value
corresponds to the control media pH registered after the sterilizing
process.
2.2. Plant material and seeding
Seeds used as plant material for testing were commercial seeds
of tomato, Lycopersicon esculentum cv. Tres Cantos (Fitó), and let-
tuce, Lactuca sativa cv. Trocadero (Vilmorin). Seeds were surface-
sterilized by soaking for 2 min. in a commercial sodium hypo-
chlorite (2%) solution to which a few drops of Tween-20 had been
added. Then they were rinsed twice in sterile distilled water and
seeded under sterile conditions into the culture tubes with MS
media containing the different chemicals. Each treatment consisted
of 20 seeds. Prior to starting the assays, samples of the same seed
batches were tested for rapid and homogeneous germination under
the assay conditions.
2.3. Plant culture and analysis
In the first experiment, cultures were incubated at 20  1  C
under a 16 h light photoperiod. To determine the short and long-
term effects of the chemicals, plant growth was periodically
monitored over time up to 90 days of culture. Germination was
recorded for the first 7 days of culture and the relative inhibition of
seed germination (GI) was calculated by the following standard
equation [5]:
Relative Germination Inhibition ð%Þ
¼ ½ðGerminationControl
Treatment Þ=GerminationControl *100
The fresh and dry weights of the plantlets were separately
recorded for shoots with the leaves and for roots. Proline, a plant
metabolite that increases under water stress conditions, i.e. in
drought, was spectrophotometrically determined at the end of the
culture in plant samples from all treatments, according to Bates
et al. [6]. The second experiment was performed and monitored as
the first one, but ended earlier, after 25 days of culture.
2.4. Statistical analysis
Germination and growth data were analyzed by one-way anal-
ysis of variance for every chemical treatment, using Statgraphics.
When significant effects were identified, LSD at P ¼ 0.05 was
applied to test for differences between the means.
 L. Martin-Closas et al. / Polymer Degradation and Stability xxx (2014) 1e7
3. Results and discussion
3.1. Changes in the culture media
The availability of the soil nutrients to plants is pH-dependent;
slightly acidic pH values optimize nutrient uptake by roots and thus
do not restrict plantlet growth. The overall choice of pH for plant
tissue culture is 5.7 before autoclaving; after cooling the culture
medium, pH decreases to 4.7e5.3 depending on the medium
composition.
In our experiment, the tissue culture system monitored was
prepared following well-known standard procedures, and the pH in
the control medium decreased as expected (Table 2). The chemicals
tested are fully soluble in water at the concentrations tested and
can be routinely incorporated to the medium. However, since we
had no knowledge of prior tissue culture media preparation with
these compounds, we followed the precautionary measure of
adding them to the media after autoclaving. In this way the changes
in the acidity of the media as affected by the nature and concen-
tration of the chemicals under study, i.e. the predictable situation a
soil or substrate may follow after being polluted with a chemical,
can be also acknowledged.
Supplementing the medium with 50 or 500 mg l
1 of the acids
tested, decreased the medium pH by 0.8e1.7 depending on the
chemical, while the 5 mg l
1 concentrations, together with the three
butanediol treatments, had no specific effects on the medium pH
(Table 2). The growth of the lettuce seedlings for 25 days contributed
to decreasing pH to 3.6e3.7 in the control medium and in the
5 mg l
1 concentrations, a usual situation for plant tissue cultures
growing in MS medium. However, in the media with 500 mg l
1
adipic and succinic acids and pH pre-adjusted, the starting pH was
lower and did not change after culture. Equally, when the pH was
adjusted after the incorporation to the medium of 500 mg l
1 adipic
and succinic acid, it remained unchanged or decreased only slightly,
respectively, 25 days after culture. This suggests independent effects
of the chemicals and pH values on plant growth.
In both field and in most bioassay tests for testing chemicals, the
pH component of the chemical usually goes together with the
compound and concentration tested. The effect of the compound is
frequently reported in soils or substrates (or even hydroponic cul-
ture systems) where the pH may range from 3.5 to 8.1. To predict
the impact of chemicals in the environment, their interactions with
the soil need to be considered, and pH is one of the important el-
ements in controlling many soil processes [7]. As pH, and particu-
larly high pH values, may create conditions in which some minerals
are unavailable for plant growth, the confounding effects of the
chemical and pH cannot be properly identified. In vitro systems
provide unique conditions where the physico-chemical properties
of the culture medium can be controlled and monitored; thus, they
offer the potential to investigate the specific effects of chemicals,
together with the associated changes they produce in the
environment.
Table 2
The pH values of the MS culture media after autoclaving and after 25 days of growth of the lettuce seedlings.
pH Control Adipic Acida
5 50 500
After autoclaving 5.1d 4.9 4.3 3.9
pH pre-adjustedb 3.7 3.7 4.0 3.9
pH post-adjustedc 3.7 3.7 3.9 5.1
a
mg l
1 in the culture medium.
b
Medium pH after 25 days of the lettuce seedling growth when adjusted prior to autoclaving.
c
Medium pH after 25 days of the lettuce seedling growth when adjusted following autoclaving and the supply of the chemicals.
d
Numbers are average of the pH medium in 5 culture tubes.
Please cite this article in press as: Martin-Closas L, et al., An in vitro crop plant ecotoxicity test for agricultural bioplastic constituents, Polymer
Degradation and Stability (2014), http://dx.doi.org/10.1016/j.polymdegradstab.2014.03.037
3
3.2. Lettuce and tomato seed germination
Several bioassays use lettuce seeds and there is a general
agreement that this species is among the most sensitive and widely
available for ecotoxicity bioassays, e.g. Refs. [8,9]. Tomato, culti-
vated with mulches and among the most important vegetable crops
in the world, was also selected for the bioassay. Germination was
previously tested in the control medium to ensure a high and ho-
mogeneous rate in this in vitro study system, and for both species it
began 2 days after the initiation of the culture. In tomato, Lyco-
persicon esculentum cv. Tres Cantos, most seeds had germinated
after 4e6 days in culture and the germination rate was over 95%,
with no differences between treatments. Germination rate of let-
tuce, Lactuca sativa cv. Trocadero, was over 95% at 7 days and was
not affected by the compounds with the exception of the
500 mg l
1 adipic acid pre-adjusted pH treatment, where only 73%
seeds germinated. The GI index for this treatment was 24%.
Adjusting the pH allowed all seeds to germinate irrespective of the
compound and concentration, including adipic acid. When low pH
values were caused by succininc acid, germination was not
inhibited; thus it can be concluded that adipic acid has some effect
on lettuce seed germination, this effect mediated only to some
extent by pH changes.
Germination is a highly conserved developmental process
following seed imbibition that involves the mobilization of the food
reserves stored in the endosperm, and ends up with the emergence
of the radicle through the seed coat. After germination, and until
the seedling becomes photosynthetically self-sufficient and the
plantlet growth phase starts, seedling growth relies on the reserves
stored in the seed. In many standardized ecotoxicity tests carried-
out in pots, the underground environment of the seed cannot be
monitored; the emergence of the radicle is not targeted and the
reports on germination rate match with a physiologically unde-
fined step after the radicle emergence, when the seedling is seen to
emerge from the substrate. This reported emergence is dependent
on specific conditions of the assay, e.g. the depth of the seeds in the
substrate. In plant tissue culture, seeds are placed on a transparent
culture medium that enables close and detailed monitoring of the
seeds and plantlets at any time along the experiment. In this me-
dium the precise effect of chemicals on seed germination, i.e. in the
emergence of the radicle, can be observed and differentiated from
other developmental processes that follow.
Simple bioassays have already proven useful in ecotoxicity
testing. Among them the germination of seeds in Petri dishes es-
tablishes the inhibition in germination and early developmental
stages of the plant species tested caused by soil contaminants that
are added to the water in the plate. However, the short running
time of the assay, the very rapid seedling growth in the limited
space of the plate, fungal or microbial contamination, and the
horizontal growth imposed to seedlings after germination are
methodological problems that result in limited information on the
effects of chemicals on plant growth. Additionally, environmental
Succinic Acida Lactic Acida 1,4-Butanediola
5 50 500 5 50 500 5 50 500
5.1 4.3 3.8 5.0 4.1 3.4 5.1 5.3 5.1
3.6 3.8 3.9 e e e e e e
3.7 3.9 4.5 e e e e e e
4 L. Martin-Closas et al. / Polymer Degradation and Stability xxx (2014) 1e7

requirements for seed germination and early plant metabolism are 0.3
not necessarily maintained along time but differ from those further *
required by the plant to growth. Not unexpectedly, inhibitory ef- 0.25
fects on plant growth have been reported for compounds proven to *

Shoot Dry Weight (g)

*
have no significance for germination [10e13]. Germination has 0.2

resulted to be a less sensitive endpoint than plantlet growth in

0
ecotoxicity testing [10,14]. 0.15
5
Germination may proceed when suitable environmental con- 50
*
ditions, in terms of water availability and temperature, are met. It is 0.1
500
basically independent from the soil composition, which is not a key *
issue until the roots become functional and contribute to the 0.05

nutrient eand pollutant- uptake. Thus, provided that the above

requirements are satisfied, germination is largely independent of 0
Control Adipic Acid Succinic Acid Butanediol Lactic Acid
soil quality. Several authors suggest that germination is easy to
Treatment
determine but it may not be a useful endpoint for plant testing [8].
0.16
This is also the case for bioassays intended for vegetable crops *
growing in mulch environments; germination and early grow are 0.14
*
not endpoints: mulches are not used with plants emerging from *
*
0.12
seeds but installed together with fully photosynthetic young plants

Root Dry Weight (g)

**
from a seedbed. 0.1

0
0.08
3.3. Plantlet growth 5
0.06 50

Tomato and lettuce plantlets were cultured and their growth
was monitored. In tomato, growth was severely restricted by the
presence of 50 and 500 mg l
1 of adipic acid in the culture medium,
which significantly reduced the shoot and specially the root dry
weight (Fig. 1). However, 5 mg l
1 of this compound caused the
opposite effect. Without prior indication of how adipic acid could
promote growth, we may speculate on its integration in the plant
metabolism to provide acetyl-CoA [15], or on a hormesis effect
which has occasionally been reported when testing very low con-
centrations of some otherwise toxic compounds in bioassays
[8,16,17]. On the other hand, tomato plantlet growth was enhanced
by butanediol and lactic acid. For the highest lactic acid concen-
tration tested, the effect was significant in the shoots, while for 5
and 50 mg l
1 the underground part of the plantlets was promoted
(Fig. 1). As a result, in all three lactic acid concentrations assayed the
final plant weight was significantly higher. All concentrations of
butanediol were effective in increasing root development.
Although no role for 1,4-butanediol is known in plant metabolism,
an isomer of this compound produced by soil bacteria has been
found to promote plant growth [18,19]. While succininc acid
treatments were ineffective in modifying the tomato plantlet
growth, all the other three compounds tested were more effective
in modifying root rather than shoot development.
Adipic acid was also strongly inhibitory of the lettuce shoot and
root growth (Fig. 2). Plantlets in the MS medium with 50 mg l
1
adipic acid grew slowly and abnormally from the beginning (Figs. 2
and 3). Irrespective of the pre or post-adjustment of pH, plantlet
growth was severely inhibited, and the increasing adipic acid
concentrations resulted in stunted leaves and progressively
reduced reddish roots that could be observed only when the pH of
the medium had been post-adjusted (Fig. 3). Other chemicals found
to inhibit germination also cause short and red (in web version)
roots [20], indicative of plant stress. Roots, the part of the plant first
to come into contact with the chemicals in the field, are sensitive to
changes in the soil environment, which may slowly modify their
growth and development. These changes go frequently unnoticed
in routine assays that analyze the germination and early growth of
terrestrial plants in pots or dishes, systems that do not allow a
close-up monitoring of the roots over time and that are strongly
dependent on the changing environmental conditions during the
tests. However, root development is critical for plant performance;
plants with limited root development are poorly competitive,
500
0.04
0.02 *
*
0
Control Adipic Acid Succinic Acid Butanediol Lactic Acid
Treatment
Fig. 1. Shoot and root dry weight of tomato plantlets grown in MS media supple-
mented with a range of concentrations (mg l
1) of adipic, succinic, or lactic acids and
1,4-butanediol. *Treatments statistically different from controls (P ¼ 0.05).
leading to smaller plants with abnormal leaf development. These
changes may result in a decrease in the commercial value of the
crop. Adipic, lactic and succinic acids have been identified in root
exudates of a variety of plant species [21] frequently grown under
protected cultivation in plastic environments, and are potential
participants of the complex plant secondary metabolism. Allelo-
pathic effects of root exudates have shown significant reduction in
the root length of strawberry at very low concentration of adipic
(30 mg l
1), lactic (9 mg l
1) and succinic (24 mg l
1) acids, but no
effects were identified on the dry weight or height of the plantlets
even at concentrations up to 60 mg l
1 [2].
Lettuce plantlets grown in a medium containing even just
5 mg l
1 adipic acid also had significantly reduced growth of shoots
and roots (Fig. 2). This differing effect of very low adipic acid,
inhibiting lettuce and promoting tomato growth, could be related
to the more vigorous growth of tomato seeds and deserves further
research. Moreover, this effect was not as obvious as with higher
doses of adipic acid and remained unnoticed during the first 3e4
weeks of culture (Fig. 3). Only when the plantlet weight was
recorded at the end of the experiment, an average reduction of over
30% was found, pointing to a long term effect of this chemical.
Studies in plant pots have shown that adipic acid limits the plant
growth only at substantially higher concentrations, while in acute
toxicity studies in Petri dishes germination has been sensitive to
30 mg l
1 adipic or over [22,23]. To our knowledge, the most sen-
sitive bioassay reported found some inhibition of root elongation at
a concentration of 15 mg l
1 [24].
The other three chemicals tested, succinic and lactic acids, and
butanediol, had no effect on final weight of the plantlet shoots
(Fig. 2). However, 500 mg l
1 lactic acid negatively affected the
early development of the plantlets (Fig. 4); yet later on the lettuce

Please cite this article in press as: Martin-Closas L, et al., An in vitro crop plant ecotoxicity test for agricultural bioplastic constituents, Polymer
Degradation and Stability (2014), http://dx.doi.org/10.1016/j.polymdegradstab.2014.03.037
 L. Martin-Closas et al. / Polymer Degradation and Stability xxx (2014) 1e7 5

180 presence of the chemicals in the medium. Proline is known as a

160
marker for osmotic stress in plants, and some authors have sug-
gested proline accumulation as an indicator for a wider range of
140
environmental stresses on plants [25,26].
Shoot Dry Weight (mg)

120 This in vitro bioassay with crop plants has identified toxicity
*
100
risks that may emerge during the biodegradation of the mulches
0
when the four monomers are released. It has been used here with
80 5
* individual chemicals as it is most frequent for other standards.
50
60
500
However, the monomers released will mix with the soil and with
* other components of the mulches (e.g. additives) in a multifaceted
40
manner very dependent on the mulch composition and on a range
20
of soil and environmental conditions (e.g. pH); the effect of the final
0 mixture will not necessarily be additive. Mulch biodegradation
Control Adipic Acid Succinic Acid Butanediol Lactic Acid
might finally affect crop plants or not depending not only on the
Treatment
individual concentration of each monomer but also on the in-
18
teractions between them, and with the soil environment. The
* bioassay has proven to be sensitive to very low concentrations of
16
chemicals and it will be of interest to investigate more complex
14 mixtures of the mulches components.
Root Dry Weight (mg)

12

10 * *
8 * *
6
* 500
4
*
2
0 term toxicity of the chemicals present in agricultural mulches on
Control Adipic Acid Succinic Acid Butanediol Lactic Acid
Treatment
Fig. 2. Shoot and root dry weight of lettuce plantlets grown in MS media supple-
mented with a range of concentrations (mg l
1) of adipic, succinic, or lactic acids and
1,4-butanediol. *Treatments statistically different from controls (P ¼ 0.05).
plants resumed growth and the final weight of the shoots was
equivalent to that of control plants (Fig. 2). On the contrary, lactic
acid proved to be the most effective in reducing root development
(Fig. 2). As reported in tomato, we found no effect of succinic acid,
and for butanediol the highest concentration tested significantly
enhanced root development (Figs. 2 and 4).
Both in tomato and in lettuce, and especially in the plantlets
grown with the highest concentration of adipic acid, all treatments
increased the plantlet proline by up to 15 fold (Fig. 5). This suggests
that proline levels are related to the stress resulting from the
0
4. Conclusion
5
50
In this paper we designed an in vitro study system advantageous
over other bioassays currently used. The plant tissue culture system
has several advantages: 1 e allows for tight control of factors that
interact with chemicals and plants, 2 e reports on short and long
vegetable crops massively cultivated in mulch environments, and 3
e allows for close monitoring of plant development at any time
along the plantlet growth, roots included. We found different but
mostly consistent sensitivities to the chemicals tested for lettuce
and tomato. Adipic and succinic acids, and to some extent buta-
nediol, caused fairly equivalent effects. The effect of lactic acid on
root development requires further investigation.
In summary, this in vitro assay allowed monitoring the effects of
the tested compounds over time and has also proved to be more
sensitive than standard short term assays, especially noticed when
low concentrations of adipic acid were tested. We are unaware of
long-term exposure tests to adipic acid in terrestrial plants as in the
present in vitro assay. Differences obtained with lettuce and tomato
were not substantial, but indicative of the need to test the precise
species targeted. We highlight the convenience of the system for
detailed monitoring of root development; roots are usually more
sensitive to the chemicals in the soil than shoots and leaves, and

Fig. 3. Lettuce plantlets grown for 25 days in MS media supplemented with increasing concentrations of adipic acid. pH was adjusted before (left) or after (right) autoclaving and
adding the chemicals.

Please cite this article in press as: Martin-Closas L, et al., An in vitro crop plant ecotoxicity test for agricultural bioplastic constituents, Polymer
Degradation and Stability (2014), http://dx.doi.org/10.1016/j.polymdegradstab.2014.03.037
6 L. Martin-Closas et al. / Polymer Degradation and Stability xxx (2014) 1e7

Fig. 4. Lettuce plantlets after 28 days of culture with different concentrations (mg l
1) of 1,4-butanediol and lactic acid.

limited root development may compromise crop yield. Proline that

deserves further research.

Acknowledgments
a) 90

80 The authors are grateful to the Spanish Ministry of Science and

Innovation, MICINN (Grant nr. AGL2008-03733/AGR) and to the
70
University of Lleida, Spain (Grant nr. UdL006197) for financial
Proline (mg/g FW)

60 support. We also thank Sandra Cervelló and Montse Roig for

50 technical assistance.
0
40 5
50 References
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