14:15 〜 14:30
[MZZ41-03] Ecotoxicological Profiling of Digestate as Biofertilizer for Land Application: The “One Health” Perspective
キーワード：Digestate、Anaerobic digestion、Ecotoxicological Profiling、One Health
1. Introduction Anaerobic digestion (AD) is a sustainable organic waste management method to produce biogas (methane). AD results in a nutrient-rich digestate used as biofertilizer in agriculture for improving crop productivity. Despite its beneficial properties in agriculture, there are limited ecotoxicological studies to explore its environmental and human health impacts. Therefore, this study evaluates digestate quality using ecotoxicological tools and matrix-based approach within the concept “One Health Perspective.
2. Materials and Methods
2.1. Sample Collection and Chemical Analyses: Digestate was obtained from mesophilic semi-continuous anaerobic digester (35 ± 1°C) located at the National Centre for Energy and Environment, University of Benin. Samples were stored at 4°C until their physicochemical, microbiological, and ecotoxicological characterization. Parameters (pH, total solids (TS) and volatile solids (VS), chemical oxygen demand, total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), and metals (Cu, Cd, Cr, Zn, Hg, Ni, Pb)) measured followed protocols established by APHA (2005).
2.2. Allium cepa Toxicity Test: A. cepa assay was performed according to modified version of Grant’s protocol (Grant, 1982). A. cepa bulbs were exposed to different concentrations (20, 40, 60, 80, and 100% v/v) of digestate effluent and incubated at 23℃ for 5 days. Root length of onions were measured and inhibition in the root’s growth was correlated with index of the degree of toxicity. The Genotoxicity test of onion per effluent was performed according to Fiskesjö, 1985 and Chauhan et al., 1986 protocols while the mitotic index and the chromosomal aberrations was performed according to Rank et al., 2002.
2.3 Fish Toxicity: Static bioassay techniques APHA (I998) was used in the determination of acute toxicity of digestate on Clarias gariepinus fingerlings. The test procedure started with range finding test and then conducted for 96 hours period with varying concentration (20, 40, 60, 80, and 100% v/v) of digestate. Safe concentration of the digestate at 96 hrs was obtained by multiplying the 96 h LC50 by a factor of 0.1 in accordance with EIFAC (1998). Toxicity factor (TF) for 24 hourly relative potency measurements of the digestate were also determined.
2.5 Earthworm Toxicity: Earthworms (Aporrectodea longa) were exposed to a mixture of soil and digestate in different concentrations (20, 40, 60, 80, and 100% v/v) according to OECD 222/2004 guidelines and Pivato et al. (2013). Earthworm behavior and biochemical (SOD CAT and GST) were monitored according to the method of Zou et al., (2012).
2.6 Microbiological Analysis: The pour plate method was used for microbial isolation and enumeration of viable microorganisms present in the digestate. From the dilutions of each sample, 0.1 ml aliquot was transferred aseptically into freshly prepared nutrient agar plates and spread evenly on the medium in duplicates. The inoculated plates were incubated at 37°C for 24 h after which, plates were examined for growth.
3. Results Digestate analyses showed that heavy metal concentrations were above permissible limits for wastewater by WHO except for Zn and Cu. Acute toxicity of fish showed gill distortion and fusion of adjacent lamella, while the liver revealed swelling of the hepatocytes with mild steatosis and fatty changes. The biochemical analysis of earthworms revealed alteration in SOD, CAT and GST. Allium cepa root tip cells showed a significant decrease in mitotic index (MI) indicating cytotoxicity and DNA damage. Also, the microbial analysis revealed bacterial isolates, Bacillus subtilis, Salmonella sp, Pseudomonas sp, Proteus sp, Enterobacter sp, Chromobacterium sp Bacillus spp. and Escherichia coli.
4. Conclusion: Findings of this study indicate that digestate effluents may constitute serious environmental and public health concerns. Therefore, further treatment of digestate is required before their exploitation in agriculture.
2. Materials and Methods
2.1. Sample Collection and Chemical Analyses: Digestate was obtained from mesophilic semi-continuous anaerobic digester (35 ± 1°C) located at the National Centre for Energy and Environment, University of Benin. Samples were stored at 4°C until their physicochemical, microbiological, and ecotoxicological characterization. Parameters (pH, total solids (TS) and volatile solids (VS), chemical oxygen demand, total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), and metals (Cu, Cd, Cr, Zn, Hg, Ni, Pb)) measured followed protocols established by APHA (2005).
2.2. Allium cepa Toxicity Test: A. cepa assay was performed according to modified version of Grant’s protocol (Grant, 1982). A. cepa bulbs were exposed to different concentrations (20, 40, 60, 80, and 100% v/v) of digestate effluent and incubated at 23℃ for 5 days. Root length of onions were measured and inhibition in the root’s growth was correlated with index of the degree of toxicity. The Genotoxicity test of onion per effluent was performed according to Fiskesjö, 1985 and Chauhan et al., 1986 protocols while the mitotic index and the chromosomal aberrations was performed according to Rank et al., 2002.
2.3 Fish Toxicity: Static bioassay techniques APHA (I998) was used in the determination of acute toxicity of digestate on Clarias gariepinus fingerlings. The test procedure started with range finding test and then conducted for 96 hours period with varying concentration (20, 40, 60, 80, and 100% v/v) of digestate. Safe concentration of the digestate at 96 hrs was obtained by multiplying the 96 h LC50 by a factor of 0.1 in accordance with EIFAC (1998). Toxicity factor (TF) for 24 hourly relative potency measurements of the digestate were also determined.
2.5 Earthworm Toxicity: Earthworms (Aporrectodea longa) were exposed to a mixture of soil and digestate in different concentrations (20, 40, 60, 80, and 100% v/v) according to OECD 222/2004 guidelines and Pivato et al. (2013). Earthworm behavior and biochemical (SOD CAT and GST) were monitored according to the method of Zou et al., (2012).
2.6 Microbiological Analysis: The pour plate method was used for microbial isolation and enumeration of viable microorganisms present in the digestate. From the dilutions of each sample, 0.1 ml aliquot was transferred aseptically into freshly prepared nutrient agar plates and spread evenly on the medium in duplicates. The inoculated plates were incubated at 37°C for 24 h after which, plates were examined for growth.
3. Results Digestate analyses showed that heavy metal concentrations were above permissible limits for wastewater by WHO except for Zn and Cu. Acute toxicity of fish showed gill distortion and fusion of adjacent lamella, while the liver revealed swelling of the hepatocytes with mild steatosis and fatty changes. The biochemical analysis of earthworms revealed alteration in SOD, CAT and GST. Allium cepa root tip cells showed a significant decrease in mitotic index (MI) indicating cytotoxicity and DNA damage. Also, the microbial analysis revealed bacterial isolates, Bacillus subtilis, Salmonella sp, Pseudomonas sp, Proteus sp, Enterobacter sp, Chromobacterium sp Bacillus spp. and Escherichia coli.
4. Conclusion: Findings of this study indicate that digestate effluents may constitute serious environmental and public health concerns. Therefore, further treatment of digestate is required before their exploitation in agriculture.