Collection and Maintenance of Earthworms:
Earthworms were collected and carefully carried within an hour of collection to the laboratory in a moist soil sample. They were placed in plastic-pots (diameter: 25cm, length: 30cm) punctured at the bottom and perimeter (for easy drainage and aeration) and packed with soil acquired from a fertile field with no history of herbicide use was utilized for this experiment. At the initial stage during acclimatization, the earthworms were fed crushed egg shells, and the soil was watered every 48 hours to field capacity; 100ml of water per pot, to avoid excessive moisture content and water logging and this was continued during the course of the experiment. After 48 hours, the worms were extracted from the pots and kept on a moist filter-paper to rid the earthworms of their intestinal contents.
The experiment was designed in line with studies conducted by Gaupp-Berghausen et al. (2015) with slight changes. A total of 120 earthworms were used for this experiment. The number of earthworms introduced per pots was according to the description of Edwards and Bohlen (1996) which suggested that an average of >300 worms occupy a square meter of soil. The earthworms were divided into 10 groups, ten earthworms per pot, three replicates and thirty earthworms per group. The antioxidant-rich plants were made available as detritus in a combination of broken egg shells and the earthworms were fed every 48hours, by mixing the feed combination with the soil. The earthworms were acclimatised for 14 days in the laboratory at room temperature (23±1°C) as described by Heimbach (1984). After acclimatization, three concentrations of glyphosate – 1, 2 and 3 percent of glyphosate, which contained glyphosate concentration of 360g/L (Hebel Enge Biotech Co. Ltd) was applied twice. The worms were divided into four groups with varying concentrations per group, Group A: Control, only food and water; Group B: 1% glyphosate + Ocimum gratissimum (Scent Leaf); Group C: 2% glyphosate + Telfairia occidentalis (Ugu Leaf) and Group D: 3% glyphosate and food alone. The experiment lasted 14 days.
Determination of Worm Weight
To determine their average weight, the earthworms were weighed together on a weighing scale. The average weights of the worms were taken and recorded at the start of the experiment. The weights were further taken on days 3, 7 and 14. They were subsequently washed with distilled water, desiccated with moist filter-paper and after weighing, introduced into each bucket. Upon release, the earthworms burrowed directly into the soil.
Oxidative Stress Assays
The earthworms were collected from each pot on days 3, 7 and 14 respectively after exposure. Afterwards, they were properly washed with distilled water and saline to eliminate residual soil contents. 0.2 grams of the earthworm tissue was cut and subsequently homogenized in a solution of 1.8ml homogenizing buffer (50mM Tris-Hcl, 1.15% KCl, pH 7.4) for the antioxidant enzyme analysis.
Lipid Peroxidation Analysis
Lipid peroxidation of the earthworm body was determined using the method of Gagne (2014). Lipid peroxidation in the formation of thio-barbituric reactive substances (TBARS) was calculated by combining 1000μl TCA (20%) and 2000μl TBA (0.675%) with 0.5 ml tissue supernatant and then heated at 100oC for 1 hour. After cooling, the precipitate was extracted for 5 minutes by centrifugation at 3500 X g and the absorbance was measured at 535 nm. The unit / mg protein lipid peroxidation was measured with a coefficient of molar-extinction of 1.54 X 105 M /cm.
Analysis of Catalase Activity
Catalase (CAT) activity was analyzed in the earthworms using the Beers and Sizer (1952) technique with a slight modification. The reaction mixture was prepared into quartz spectrophotometer cuvette. H2O2 was prepared with PO4 buffer; 100μl of tissue homogenate was added to 1.9 ml of 30mM of H2O2 substrate. The absorbance of the sample was read at time zero (A0) and after 1 minute (A1) at 240 nm.
Analysis of Superoxide-dismutase (SOD) Activity
The analysis of Superoxide dismutase (SOD) in earthworms was done following the method of Valeska et al. (2009). The function of superoxide-dismutase was analyzed by its potential in the inhibition of epinephrine auto-oxygenation. 2.95 ml of 0.05 M Na2CO3 buffer pH 10.2, 0.02 ml of tissue supernatant and 0.03 ml of epinephrine in 0.0025 N HCl were used in the reaction mixture (3ml) to start the reaction. The reference cuvette had a buffer of 2.95 ml, a substrate of 0.03 ml (epinephrine) and 0.02 ml of H2O. By measuring the absorbance shift at 480 nm for 60 seconds, enzyme activity was calculated.
Analysis of Glutathione (GSH) Concentration
GSH analysis was conducted in according to the method of Blume et al. (1975). In a test tube, 500 μL of homogenized tissue was pipetted into 4000 μL of 0.08N sulphuric acid (H2SO4) and carefully mixed and left for 10 mins at room temperature. 500 μL of tungstate mixture was then pipetted to the precipitated protein. The bottle was covered and the solution was agitated thoroughly for 300 seconds. The mixture was kept for 120 seconds at 20-22OC. Then, the solution was centrifuged at 860 rpm for 20 minutes. In 2500 μL of tris-buffer, two millilitres of the clear extract were pipetted, then 200 μL of DNTB reagent was applied. By using 2000 μL of water instead of homogenized tissue, a reagent blank was also prepared. The colour was produced after 30-60 seconds and the optical-density was calculated at 412 nm.
DNA Fragmentation Assay
DNA fragmentation analysis was carried out according to Ibrahim et al. (2013). The earthworms were collected per pot on days 3, 7 and 14 after exposure. Afterwards, they were properly rinsed with distilled water and saline to remove residual soil contents and weighed. 100 milligrams of the earthworm tissue was cut, and subsequently digested in a solution of 1000 μL buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, and 0.5% Triton × 100). Then the solution containing the earthworm tissues were incubated in an ecotherm at 56°C for 12 to 18hr in tightly capped tubes. After which, the remaining unused tissues were stored in a saline solution and refrigerated to preserve the harvested earthworm tissue for further analysis.
Tissues are lysed in 1000 μL buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, and 0.5% Triton × 100). 500 μL of 25 percent TCA was pipetted separately into tubes containing the complete DNA pellets (marked P) and the smaller particles of DNA supernatants (marked S). Both tubes are allowed to sit overnight at 4°C, and precipitated DNA is acquired through centrifugation. Each tube is further treated with 80 μL of 5% TCA, afterwards a heat treatment at 90°C for 15 min is implemented. 1000 μL newly prepared diphenylamine (DPA) reagent is treated differently into each sample, tubes and are allowed to stay overnight at 20-22oC, and optical density is set at 600 nm. DNA fragmentation was calculated as follows:
% DNA fragmentation = [S/ (S + P)] * 100 where S is Supernatant and P is Pellet. The intensity of extracted DNA was also calculated using Image J software.
Statistical analysis was conducted using GraphPad Prism version (8.0.2) for windows. Mean values of the parameters were compared between the exposed earthworm groups and the control group using One-way ANOVA. P-value less than (<) 0.05 was considered to be statistically significant.