Research article
Phytotoxicity assay to assess sewage sludge phytoremediation rate using guaiacol peroxidase activity (GPX): A comparison of four growth substrates

https://doi.org/10.1016/j.jenvman.2020.110413Get rights and content

Highlights

  • Sewage sludge amendment dose determination in phytoremediation by GPX measurement.

  • Guaiacol peroxidase activity (GPX) is a reliable indicator of toxicity.

  • Soil water-based extracts agar solidified are good toxicity test medium.

  • The GPX activity test in plants need “in situ” evaluation.

  • Chronic toxicity measurement on agar medium may not reflect natural conditions.

Abstract

Waste disposal such as sewage sludge (biosolids) in phytoremediation is a sustainable remediation alternative for fertilizers, therefore there is a need to develop a test that will allow to determine phytoremediation dose of biosolids from the best-for-plant-growth point of view. In order to determine the doses of biosolids to degraded soils, tests based on germination of seeds and root elongation are commonly used, but also, they are subjected to large errors caused by low repeatability of results and differentiation. That is why there is a need to introduce new testing solutions that will be of use based on more reliable indicators such as biochemical activity of selected plant enzymes. The aim of the study was to demonstrate high efficiency of the guaiacol peroxidase activity (GPX) in plant-based toxicity tests used as an optimal dose amendments indicator in heavy metal degraded soil phytoremediation process. GPX were measured in underground and above ground parts of Sinapis alba L. and Brassica rapa L. in relation to germination index (GI) and biomass cultivated on four different substrates (raw degraded soil, sterilized degraded soil, water extract from degraded soil solidified with agar, water extract from degraded soil solidified with Murashige-Skoog medium). Each testing soil substrate was enriched with sewage sludge (food industry origin) in the percentage share (w/w) of 5, 10, 15, 20 and 25. The process was carried out under controlled conditions of the phytotronic chamber for a period of 14 days. The obtained values were compared for each plant separately and for all substrates and amendments rates of sewage sludge. GPX activity was expressed as a percentage increase or decrease in relation to the GPX in soil substrates without addition of sewage sludge which allowed to determine their positive or negative impact on substrate toxicity. Results of GPX activity showed that the water-based soil extracts solidified with agar give more accurate results in relation to the tests on raw soil. It has been demonstrated that the optimal phytoremediation dose of sewage sludge was in the range of 15–20%, with values of 5% and 25% respectively favoring or inhibiting plant development expressed in GPX activity. The most differentiating GPX values were obtained for the roots.Measurement of GPX activity in the roots of Sinapis alba L. cultivated on soil agar-based tests is a good, new and easy additional or alternative to the old tests based on germination and increase biomass measuring as an indicator in the assessment of optimal phytoremediation sewage sludge.

Introduction

The constantly expanding areas of contaminated or degraded soils that are not suitable for agricultural production and very often for any plant's vegetation are still of concern. That why it is important to search for new sustainable phytoremediation varieties on such soils (energy crop cultivation) with the use of surging residual waste, i.e. sewage sludge or biosolids (Fijalkowski et al., 2018). It is essential to use some kinds of fertilizers/enhancers on degraded terrains because of their low plant-vegetation capabilities (lack of nutrients and organic matter). One of them is sewage sludge which has huge phytoremediation potential (Kapanen et al., 2013) and can successfully replace mineral fertilizers giving additional benefits in the form of increased carbon sequestration (Finnan and Styles, 2013) and their environmental use is highly related with new directions of management in recycling energy and materials shown in article 14 of Council Directive 91/271/EEC where states that sewage sludge has to be reused in each appropriate case which is a result of European Commission eco-innovation trend related to the main concept of “reduce, reuse, recycle” (Kacprzak et al., 2017).

Due to the fact that sewage sludge is and will continue to be generated, it is very important to use their remediation potential as an alternative to expensive combustion technologies or unfavorable storage, which is also compatible with circular economy and sustainable development (Fijalkowski et al., 2017). Although the characteristics of sewage sludge is variable and depends on the source and process of wastewater treatment i.e new treatment technologies based on algae-bacteria consortia (Sepehri et al., 2020) produce sludge characterized by large volume of algae among bacteria which may be an additional source of assimilable forms carbon and nitrogen (Sepehri and Sarrafzadeh, 2018) - nevertheless it is a material rich in organic matter and essential nutrients for plants and microorganisms (Christofoletti et al., 2012).

Due to the changing composition of the sewage sludge, it is very important to pre-determine their dose each time of use - because have to be combined the properties of soil (Oleszczuk and Hollert, 2011) with the nature of sewage sludge (Kapanen et al., 2013). For this purpose, various types of commercially available toxicity tests or adapted from the literature may be used. To check the suitability of the analytical method, parameters such as accuracy, sensitivity, reproducibility, simplicity, cost-effectiveness, flexibility and speed must be taken into consideration (Wieczerzak et al., 2016). The use of toxicity tests as analytical tools to determine phytoremediation dose of sewage sludge requires modification of existing methods to meet most of the above-mentioned requirements. Due to the nature of sediments, the selection of a method determining their toxicity in soil and the phytoremediation process is very important also with reducing the toxicity of degraded soil while ensuring the best possible plant-growing conditions.

Therefore, primarily “by-plant” tests are commonly used where the indicator organism is seed developing and/or early plant development. The next step is to choose the measurable parameters of the plant's response to environmental conditions that will be directly related to its development potential. As a standard, such parameters include: germination index (GI) (Luo et al., 2018), root length measurement and biomass weight (Gruiz et al., 2016), and tests are carried out directly on soil substrates (Oleszczuk, 2009). Very often, the substrate used for testing is filter paper; however, it turns out that the effect of the paper, e.g. on the adsorption of heavy metals, may itself distort the plant's response (Di Salvatore et al., 2008). Therefore, attempts were made to increase the sensitivity of the tests by replacing filter paper with other materials such as polyvinyl (Takijima, 1960), chemically inert seed tray (Wang, 1993)and others. However, due to the simplicity, speed and economy of the test, filter paper is widely used (Di Salvatore et al., 2008).

Seed germination index (GI) was first proposed in 1981 which used Lepidium sativum seeds in a germination test to assess the toxicity of compost. However, this parameter is burdened with a large error due to the high uniqueness of results, and the same characteristics of the seed, primarily that seed germination is an accidental event (Luo et al., 2018). Currently, the main problem of research on seed germination test is that there is no commonly accepted procedure that solves the problem of results differences(Luo et al., 2018) despite the fact that some EU countries, i.e. Italy oblige in their legislation to use GI-based tests (Cesaro et al., 2015). Oleszczuk and Hollert (2011)reports that the germination index is a weak parameter when used in sewage sludge. Weitbrecht et al. (2011) pointed out that if the incubation time is too long, the length of the root will not accurately reflect the toxicity. This time depends on the specific environmental conditions and seed species. Although this disadvantage can be overcome, it is reasonable to look for other indicator parameters based not on physical manifestation, but biochemical measurement and this unique potential have measurement of activity of the selected plant enzymes.

Stressful abiotic conditions often cause an increase in antioxidant defense mechanisms in plants. Such conditions include exposure to high or low temperatures, drought, lack of nutrients, and the presence of toxic chemicals i.e peticides (Kyriakopoulos and Doulia, 2006) and heavy metals (Kacprzak et al., 2014). Oxidative stress caused by environmental conditions contributes to the production of highly reactive compounds, i.e. reactive oxygen species (ROS) that are toxic to the plant cell itself (van Doorn and Ketsa, 2014). Plants, however, have developed a set of enzymes involved in the ROS detoxification process that include at least 300 genes (Gechev et al., 2006). To lower the ROS concentration resulting from the impact of environmental conditions (i.e. pollution, fertilizing), the plant has two ways of action, one of which is the enzymatic transformation of ROS into water, in which enzymes such as catalase (CAT) and class III peroxidases (POD - EC 1.11.1.7) are involved (Mathé et al., 2010). PODs are involved in a number of functions including coping with physical stress, pathogens or auxin and lignin metabolism (Marjamaa et al., 2009). One group of PODs of particular interest is guaiacol peroxidase (GPX - EC 1.11.1.7) which has high activity with guaiacol as a substrate. To estimate its activity it is possible to monitor the formation of guaiacol tetraguaiacol in it (van Doorn and Ketsa, 2014). Due to the fact that GPX plays a key role in response to environmental stress and can be easily determined by the colorimetric method, it can be a good new parameter (based on a different mechanism than measurement of root elongation) in assessing the response of the plant to environmental stress (Gallie, 2013). This is also confirmed by other studies, where GPX has been observed to be sensitive to changes in environmental conditions for such plants as coffee (Queiroz et al., 1998), cucumber (Lee and Lee, 2000), corn and rice (Kang and Saltveit, 2002).

The innovation of the proposed solution is the introduction of an additional GPX parameter to the traditional plant-based toxicity test. This indicator takes into account the aspect of plant stress at the cellular level. This supplementation seems advisable due to the fact that the traditional test is subject to a large error and is not useful for assessing chronic toxicity.

In general, improvement of the testing procedure and the search for new methods to increase the reliability and repeatability of seed toxicity tests is needed especially in the area of using sewage sludge or other biosolids.

The methodology proposed by the authors (Fig. 1) is based primarily on the creation of a new test in which an important novelty is the use of mediums based on solidified agar-water extract from the tested soil mixtures with sewage sludge - in relation to standard sowing – and measuring GPX activity in roots and leaves - in relation to GI germination index- and root biomass increment. The test is aimed at demonstrating the optimal dose of the sewage sludge under investigation in the remediation of heavy metal contaminated soils which in themselves are very difficult for plant vegetation in the environment. This approach will improve the use of sewage sludge and other biosolids as well as composts, which is important in the circular management of these wastes.

Section snippets

Substrates characterization

Soil used in the experiment was taken from degraded and heavy metals contaminated terrain near zinc and lead smelter (MiasteczkoSlaske, Silesia region, Poland, GPS: 50°30′31″N, 18°56′21″E). As shown in Table 1 this soil is strongly acidified has low sorption capacity and low-capacity buffer occurs. They are characterized by low fertility and organic matter productivity.

The soil samples were collected using a soil auger from the plough layer (0–30 cm) of an experimental plot without any crop. In

Seed germination

Germination percentage is shown in Fig. 2, Fig. 3. Addition of sewage sludge from 10% up have a positive effect on seeds germination. This phenomenon occurred in all growth mediums: MS, Agar medium, soil and sterile soil. The highest GI values were obtained for agar medium, next for unsterile soil and the lowest for sterile soil. This indicates that microflora present in the soil play an important role in seed germination. On 10% sewage sludge addition (s10) showed 100% germination in seeds

Discussion

It is known that heavy metals in soil cause abiotic stresses affecting plant productivity and development.

Conclusion

Two plant species: Sinapis alba and Brassica rapa were tested to assess an ecotoxicity in degraded soils contaminated by heavy metals. The measurement endpoints used were seed germination, guaiacol peroxidase activity (GPX) and protein content for both shoots and roots of seedlings. Seed germination was reported to be less sensitive endpoint and it is often inconclusive as toxicity indicator. Germination, biomass growth and development were adversely affected by contaminated soil and applied

CRediT authorship contribution statement

Anna Kwarciak-Kozlowska: Writing - review & editing, Conceptualization.

Acknowledgements

The scientific research was funded by the statute subvention of Czestochowa University of Technology, Faculty of Infrastructure and Environment. The presentation of results in this research has been financed from the EnviSafeBioC project - contract No. PPI/APM/2018/1/00029/U/001 - the project is financed by the Polish National Agency for Academic Exchange NAWA.

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