results and those from the acute toxicity study (see Sect. 5) demonstrate that this
process can be effectively used to treat wastewater from pharmaceutical industry.
5 Oxidative Stress Determination Prior and Posttreatment
Using Hyalella azteca as Biomarker
5.1 Procurement, Culturing, and Maintenance of Specimens
Hyalella azteca was collected from its natural habitat in San Miguel de Almaya
Lake, municipality of Capulhuac (State of Mexico), and transported to the laboratory
under constant aeration in plastic bags. Breeding stock was transported to the
laboratory using the water source in which the organisms were reared. Water used
for transporting organisms was well oxygenated (90–100% saturated). Upon arrival
at the testing laboratory, the organisms were gradually acclimated to the laboratory
holding and testing conditions so they would not get stressed. Test organisms were
in good health, and the mortality rate for juvenile Hyalella did not exceed 20%
[47]. The collected organisms were morphologically identified [48]. To eliminate
potential differences in sensitivity to contaminants due to acclimation to local
conditions or maternal effects, we used organisms from the same clade that had
been cultured under the same feeding conditions, temperature, and photoperiod for
approximately 4 months (third-generation neonates obtained by sexual reproduction). During culture, specimens were maintained in reconstituted water
(NaHCO 3 ¼ 174 mg L
À1 ; MgSO 4 ¼ 120 mg L
À1 ; KCl ¼ 8 mg L
À1 ; and
CaSO 4 .2H 2 O ¼ 120 mg L
À1 ; all reagents were obtained from Sigma-Aldrich,
St. Louis MO), pH 7.5–8.5, room temperature with constant oxygen
(6.4–6.6 mg L
À1 , O 2 ), and 12 h/12 h light/dark photoperiod and were fed ground
lettuce ad libitum. The estimated number of surviving adults and the production of
young in each culture chamber, dates of culture renewals, numbers and age classes of
transferred individuals, daily feedings, and water quality measurements were
documented.
5.2 Artificial Sediment
The employed artificial sediment was 70% sand (0.2 mm), 20% kaolinite
(<0.002 mm), and 10% organic matter (0.2 mm). The organic matter source was
lamb compost inactivated by dry heating at 55–60
C for 3 days. The sediment was
sterilized with three 15-min autoclave cycles at 121
C and 15-lb pressure, separated
by 1-h interval [49, 50].
254
R. Natividad et al.
process can be effectively used to treat wastewater from pharmaceutical industry.
5 Oxidative Stress Determination Prior and Posttreatment
Using Hyalella azteca as Biomarker
5.1 Procurement, Culturing, and Maintenance of Specimens
Hyalella azteca was collected from its natural habitat in San Miguel de Almaya
Lake, municipality of Capulhuac (State of Mexico), and transported to the laboratory
under constant aeration in plastic bags. Breeding stock was transported to the
laboratory using the water source in which the organisms were reared. Water used
for transporting organisms was well oxygenated (90–100% saturated). Upon arrival
at the testing laboratory, the organisms were gradually acclimated to the laboratory
holding and testing conditions so they would not get stressed. Test organisms were
in good health, and the mortality rate for juvenile Hyalella did not exceed 20%
[47]. The collected organisms were morphologically identified [48]. To eliminate
potential differences in sensitivity to contaminants due to acclimation to local
conditions or maternal effects, we used organisms from the same clade that had
been cultured under the same feeding conditions, temperature, and photoperiod for
approximately 4 months (third-generation neonates obtained by sexual reproduction). During culture, specimens were maintained in reconstituted water
(NaHCO 3 ¼ 174 mg L
À1 ; MgSO 4 ¼ 120 mg L
À1 ; KCl ¼ 8 mg L
À1 ; and
CaSO 4 .2H 2 O ¼ 120 mg L
À1 ; all reagents were obtained from Sigma-Aldrich,
St. Louis MO), pH 7.5–8.5, room temperature with constant oxygen
(6.4–6.6 mg L
À1 , O 2 ), and 12 h/12 h light/dark photoperiod and were fed ground
lettuce ad libitum. The estimated number of surviving adults and the production of
young in each culture chamber, dates of culture renewals, numbers and age classes of
transferred individuals, daily feedings, and water quality measurements were
documented.
5.2 Artificial Sediment
The employed artificial sediment was 70% sand (0.2 mm), 20% kaolinite
(<0.002 mm), and 10% organic matter (0.2 mm). The organic matter source was
lamb compost inactivated by dry heating at 55–60
C for 3 days. The sediment was
sterilized with three 15-min autoclave cycles at 121
C and 15-lb pressure, separated
by 1-h interval [49, 50].
254
R. Natividad et al.