**5.7 Maintenance of cultures**

The media should be replaced weekly; it is not necessary if massive media (100 L) is used. Approximately 30 Daphnias per culture should be selected every week; to retrieve them a glass or plastic Pasteur pipette must be used. This activity is suggested to do at the same time when the medium is changed, this system will avoid cross-linking (American Public Health Association, 2002).

### **5.8 Selection of organisms for test**

The examination should initiate with the second or third offspring, selecting neonates with ≤ 24 birth hours. Before the initiation any test, 30 to 40 young Daphnias should be isolated on a 400 mL beaker with 300 mL of algae mix (*Ankistrodesmus falcatus, Selenastrun capricornutum,* and *Clorella*). Do not use ephippiums for Daphnia cultures, because its presence is an indicator of unfavorable conditions (American Public Health Association, 2002).
