INTRODUCTION

Soilless production systems are a form of intensive agriculture that relies on the use of growing media, which have the advantages of maximizing crop yield and product quality while reducing the use of phytopharmaceutical products (PPPs) particularly for soil disinfection (^{Asaduzzaman et al., 2015}; ^{Atzori et al., 2021}). Organic substrates (OS) growing media offer several advantages in comparison with inorganic ones (e.g., nutrient efficiency, cost, end-of-life environmental impact), so their use have been continuously increasing (^{Raviv, 2011}). Nevertheless, end-of-life (EL) OS disposal can still pose an environmental risk due to their potential to accumulate fertilizers, PPPs and salts.

Many studies have focused on promoting agricultural waste recycling, especially regarding crops residues and the production of more eco-friendly substrates (^{Diacono et al., 2019}). Even so, further research is still needed to promote the valorisation of agricultural wastes and to produce value-added products (^{O’Connor et al., 2020}). Currently, there is still a lack of proper solutions to EL-OS.

In fact, several farmers already perform the incorporation of EL-OS in agricultural soils considering that this practice increases the organic matter content of degraded soils, also contributing to fertilizing the soil (data not shown) (Figure 1).

Figure 1 End-of-life substrate accumulated in open field before incorporation in soil. 

To create adequate solutions and/or validate the use of EL-OS as a soil amendment, it is critical to evaluate the environmental safety of these residues. Therefore, the aim of this study was to assess the potential ecotoxicity of agricultural soils amended with different percentages EL-OS.

MATERIAL AND METHODS

An EL-OS, originally consisting of 100% coconut fibre, used in a commercial greenhouse for tomato soilless production, was collected at the Centre Region of Portugal. The substrate was dried and sieved and the fraction <4 mm was incorporated (2.5, 5 and 10%) in a natural agricultural soil (NAS) collected at Vairão Campus of the Faculty of Sciences, University of Porto. All amended soils (NAS-EL-OS) were left to stabilize for 48h before being used and assays were performed using standard methods. In all ecotoxicity assays, EL-OS-free, NAS was used as control.

NAS, OS and NAS-EL-OS pH and electrical conductivity (EC) were measured in a soil:water suspension (1:5 w/v) according to ISO (2021). Similarly, maximum water holding capacity (WHC_max) was determined following OECD (2016). Lastly, soil organic matter content (SOM) was measured by loss on ignition at 450°C for 8h.

Four seedling emergence and seedling growth tests were performed following ISO (2012a). Two dicotyledonous Lactuca sativa and Solanum lycopersicum and two monocotyledonous Avena sativa and Zea mays species were used as test plants. All seeds were purchased at a local supplier. Briefly, five pots were prepared for each percentage of NAS-EL-OS, including controls, and moisture was adjusted to 45% of WHC_max. Then, twenty seeds were sow per replicate. Pots were kept under optimal conditions of light and temperature (20 ± 2ºC; 16h^L:8^D) and moisture was continuously monitored and adjusted. After 50% of the control group plants emerged, exposure lasted 14 days. At the end of the assay, seed emergence was recorded, plants were harvested, fresh and dry shoot weight were determined.

Eisenia fetida reproduction test was carried out accordingly to ISO (2012b). Four containers for each percentage of NAS-EL-OS, including controls, were prepared and moisture was adjusted to 60% of WHC_max. Ten adult earthworms, originated from synchronised laboratory culture, with well-developed clitellum, were randomly distributed for each container and kept under optimal conditions of light (16h^L:8^D) and temperature (20 ± 2ºC). Once a week, moisture content was adjusted and worms were fed. After 28 days, adults were removed, counted, weighed and the containers kept at the same experimental conditions for another additional 28 days. In the end of this period, juveniles were counted.

To assess the effects on soil enzymatic activity, three containers for each percentage NAS-EL-OS were incubated for 28 days at 20 ± 2ºC. Photoperiod was set for 16h^L:8^D and moisture was kept at 80% of WHC_max. (n=3, including control group). In the end of incubation, the fraction <2mm was stored at -20ºC until further analyses. The activity of dehydrogenase (DHA), acid phosphatase (AP), urease (UR), cellulase (CL), nitrogen mineralization (NM) and potential nitrification (PN) were measured based on adaptations of ^{Schinner et al. (1996}) methodology as described by ^{Bouguerra et al. (2022}).

Statistical analyses were performed in IBM® SPPS® 26 Statistics software (IBM Corp Armonk, NY). Significant differences were calculated using one-way ANOVA followed by post-hoc comparisons using Dunnett test. Significant differences were accepted when p<0.05.

RESULTS AND DISCUSSION

NAS-EL-OS resulted in a decrease of pH values and increase in EC to a maximum value of 0.426 dS/m. Furthermore, WHC and SOM increased as the percentages of EL-OS increased in soil.

Regarding seedling growth and seedling emergence, none of the tests revelled valuable toxic effects for these endpoints. Moreover, L. sativa and S. lycopersicum showed to benefit with some of the tested percentages of EL-OS, increasing wet and dry shoot weight.

E. fetida reproduction was not affected by any of the tested percentages of EL-OS incorporation.

When compared with control (NAS) the activity of DHA, AP, and PN showed a tendency to increase with the increase of the percentage of EL-OS incorporated. Contrariwise, UR and CL activities tended to decrease. NM activity did not change compared with the control.

Physical-chemical parameters results were expectable. pH and EC are a direct consequence of the intensive use of fertilizers required in these systems being extensively explained in literature (^{Robson, 2012}). Although acidity and salinity can compromise crops quality and yield, the obtained values were within acceptable range for agriculture production (^{Van Beek et al., 2010}).

Coconut fibre OS is recognized to have an apparent low density and high WHC and SOM (^{Louro & Reis, 2020}). As expected, our results showed that the incorporation of NAS with EL-OS resulted in an increase of these parameters. Such alterations have the potential to improve aeration, WHC_max., function and fertility of soils, especially in more degraded ones.

Improvements observed on plants biomass, although significantly positives, were not detected in all species, probably indicating that the bioavailability of the nutrients on the organic matter can be enhanced. Composting is well established solution to increase organic wastes fertilizing capacity (^{Zhang et al., 2019}), possibly being a suitable approach for EL-OS. More importantly, plants and earthworms were not negatively affected, indicating that soils amended with EL-OS at the doses tested in this study were not toxic.

EL-OS amended soils increased the overall microbial metabolic activity as showed by the higher DHA activity. This can result of the increase of SOM content since it offers more substrate to support higher microbial biomass (^{Wolińska & Stępniewska, 2012}). However, enzymatic activities translate complex relations between biotic and abiotic factors. Also, the C, N, P and K content and bioavailability that EL-OS can add to soil are important factors to interpret our results regarding nitrification and mineralization processes, as well as CL and UR activities. Overall, the quantified values obtained for EL-OS amended soils did not suggest major concerns regarding their safety.

CONCLUSIONS

Our results showed that soil amended with end-of-life coconut fiber substrate (up to 10%) obtained from tomato cultivation was safe for terrestrial matrices. Nevertheless, these results should be interpreted with caution because they may be dependent of the substrate origin (farming-specific differences), agronomic practices during cultivation, the OS incorporation rate and the type of soil. Different substrates constituents, crops cultivated, certification processes and countries regulations and policies are all factors that should be consider before validating the incorporation of end-of-life substrate in open fields. Further studies are needed, not only with substrates from different provenience but also to assess the safety for aquatic resources and organisms.