Dilutions and assays were performed in incubator wells at
15 ± 0.5 ◦C. Color correction, by measuring testing sample and dilutions absorbance at 490 nm (Ashworth et al., 2010) was not applied
while the absorbance values of all samples tested was negligible.
Toxicity estimations were obtained after 15 min exposure and
expressed as EC50 value, at which 50% loss of luminescence is
obtained. EC20 values (sample concentration that gives 20% luminescence inhibition) were also calculated, as described below.
Effect estimation includes the correction factor (Rt) which is
the fraction obtained when the light output of the Control (Blank)
remaining after time t (It) is divided by the initial light output of
the untreated Control (Blank) (I0):
Rt = It
I0
% Bioluminescence inhibition = 100 × Ec − Ei
Ec
where Ec is the light emission of control and Ei is the light emission
of sample.
% Effectt = 1 +GtGt × 100
where (G) value is the ratio ofthe light lost at time (t) for a given time
t to the light remaining at time (t) for a given sample concentration.
Additionally, OMNI software provided the linear correlation of
Gamma (G) values with sample concentration. The relationship
between the concentration of a toxic material and the response
of a susceptible organism, when the response is measured in terms
of Gamma (G) values, can be described by a linear equation for the
prediction of concentration from Gamma values:
log C = b log G + log a
This equation describes a line with a slope of b and an intercept
oflog a, in which C represents concentration and G the corresponding Gamma value. EC20 concentrations were calculated by placing
G = 0.250 (effect 20%) in correlation equations. As, according to the
ISO 11348 (1998) guidelines, a toxic sample shows an effect higher
than 20%, the above values are the maximum concentrations for
which a sample can be considered as non-toxic. In case ofinfusions
a reference volume of 200 mL was assumed, which is the average
cup volume, and calculated the corresponding dry plant mass and
compared to the commercial decoction portions that are available
in markets which contain usually 2 g of dry plant mass. Referring
to their use in nutrition, the corresponding dry plant masses for
oregano, which is used as raw herb in salads it was calculated
assuming a reference volume of 500 mL on treatment (b). In order
to approach the use of herbs in cooking processes, the reference
volume was assumed to be (5 L) and evaluated on treatment (a) for
oregano and sage, respectively