2.9. Lipid peroxidation
The thiobarbituric acid-reactive substances (TBARS) method,
according to Khan and Panda (2008), with modifications by Mitton
et al. (2014), was used to estimate lipid peroxidation (LPO) in plants
grown in spiked and control (non-spiked) soils. Tissues were homogenized
(1:5) in 0.1% trichloroacetic acid (TCA). An extract
(41.2 mL) was added to a reaction mixture consisting of 150 mL of
20% acetic acid, 150 mL of 0.8% thiobarbituric acid, 50 mL of Milli Q
water and 20 mL of 8.1% sodium dodecyl sulfate (SDS). The samples
were heated at 95 C for 30 min and, after cooling for 10 min,100 mL
of Milli Q water and 500 mL of n-butanol were added. The organic
phase (150 mL) was obtained by centrifugation at 3000 g at 15 C for
10 min, and the fluorescence was registered in a microplate reader
(excitation: 520 nm; emission: 580 nm). Tetramethoxypropane
(TMP, Across Organics) was employed as an external standard and
the LPO levels were expressed as nanomoles of TMP per gram of
wet tissue.