After 24 h, animals were retrieved from the chambers. Surviving
animals from each of the four chambers holding groups of 20
individuals were pooled in four eppendorf tubes and immediately
frozen in liquid N2 and kept at −80 ◦C until further enzyme analysis.
Within 1 h of exposure five surviving juveniles from chambers
holding groups of 10 animalswere placed into 60ml screw-capped
glass jars containing 50 ml of ASTM hard water, with Chlorella
vulgaris (Beijerink, strain CCAP C211/12) at a concentration of
5×105 cells/ml, and allowed to feed for 4 h (Mc William and Baird,
2002b). Three jars containing no animals were used to establish
initial algal densities. Post-feeding experiments were conducted
in darkness to avoid algal growth and under relatively constant
temperature conditions (20±2 ◦C) provided by a thermostatted
chamber transported inside the sampling vehicle. Individual feeding
rates (cells individual−1 h−1) were determined as the change in
cell density during 4 h according to themethod given byMcWilliam
and Baird (2002b). Cell density was estimated from absorbance
measurements at = 650nm in a dual-beam spectrophotometer
(Uvikon 941) using standard calibration curves based on at least
20 data points, with an r2 > 0.98.
After 24 h, animals were retrieved from the chambers. Surviving
animals from each of the four chambers holding groups of 20
individuals were pooled in four eppendorf tubes and immediately
frozen in liquid N2 and kept at −80 ◦C until further enzyme analysis.
Within 1 h of exposure five surviving juveniles from chambers
holding groups of 10 animalswere placed into 60ml screw-capped
glass jars containing 50 ml of ASTM hard water, with Chlorella
vulgaris (Beijerink, strain CCAP C211/12) at a concentration of
5×105 cells/ml, and allowed to feed for 4 h (Mc William and Baird,
2002b). Three jars containing no animals were used to establish
initial algal densities. Post-feeding experiments were conducted
in darkness to avoid algal growth and under relatively constant
temperature conditions (20±2 ◦C) provided by a thermostatted
chamber transported inside the sampling vehicle. Individual feeding
rates (cells individual−1 h−1) were determined as the change in
cell density during 4 h according to themethod given byMcWilliam
and Baird (2002b). Cell density was estimated from absorbance
measurements at = 650nm in a dual-beam spectrophotometer
(Uvikon 941) using standard calibration curves based on at least
20 data points, with an r2 > 0.98.
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