**4. Production of cladocerans**

To keep a *Daphnia sp* production, it is required a basic laboratory infrastructure, expecting to have materials, equipment, reactive substances and organic strains such as algae and cladocerans. Daphnia has been used to evaluate chronicle and severe chemicals effects, and they are very sensitive to toxic substances. Breeding Daphnia allows to clone establishment with few genetic variation and reproductive results (American Public HealthAssociation, 2002).

#### **4.1 Laboratory infrastructure**

Laboratories should contain the minimal infrastructure necessary to develop controlled culture of micro-algae and to maintain cladocerans strains, which will be used for nutritional tests.

## **4.1.1 Materials**

246 Aquaculture

Fig. 2. Daphnia reproductive strategies. Female adults can reproduce on three different types of offspring, depending on the environmental conditions. Diploid eggs (emergent) reproduce non-sexually, develop in females and males. Haploid eggs are reproduced based on an exact chemical signal from the environment, Retrieve from (Thorp & Covich, 2001)

HealthAssociation [APHA], 2002).

**4. Production of cladocerans** 

2002).

general appearance of the released juveniles is similar to the appearance of the adult, obviously with a minor size (Mitchell et al., 2004 as cited in American Public

To keep a *Daphnia sp* production, it is required a basic laboratory infrastructure, expecting to have materials, equipment, reactive substances and organic strains such as algae and cladocerans. Daphnia has been used to evaluate chronicle and severe chemicals effects, and they are very sensitive to toxic substances. Breeding Daphnia allows to clone establishment with few genetic variation and reproductive results (American Public HealthAssociation, The containers used to grow the cladocerans reproducers should be made of the boronsilicate glass, adequate volume, depending on the production protocol that is used. They must be submitted to the appropriate cleaning routine and disinfection. To achieve these tests, enough and adequate glassware and minor equipment will be used to guarantee safe and repeatable proceedings, equipment like automatic calibrated pipettes, gagged glassware with the required capacity, stainless steel spatulas, etc.

### **4.1.2 Equipment**

The essential equipment required is: analytical scale, optical microscope, stereomicroscope, autoclave (propane or electric), sterilized air or laminar flow hood for microbiological work, potentiometer, oximeter, salinometer, conductivimeter and spectrophotometer. It will be best to have a bioclimatic chamber for temperature control, lighting and photoperiods.

#### **4.1.3 Reactives**

Is necessary to have the indispensable reactives to prepare the dilution water (every time that cladoceran *Daphnia magna* production is made, it is essential to prepare the dilution water with the hardness specification required) (American Public Health Association, 2002), and culture medium, not only cladocerans but also for micro-algae. The compound should be analytical grade and free of contamination, as heavy metals. It is suggested the known commercial brands.

On each case it is suggested to prepare concentrated solutions (standard) and from this build up the culture medium and the dilution water. It must have a control over the proceedings to prepare the standard solutions and the culture medium and special attention on preserving substances that may be able to degrade or contaminate (vitamin solutions for example); in all cases it is necessary to apply the maintenance conditions to avoid the alteration on chemical concentration from the standard solutions.

#### **5. Organisms for testing**

Daphnia is widely distributed, this make it easy to obtain not only in laboratories but also in research institutions. 20 to 30 organisms are plenty to start the production. Additionally the reproduction of Daphnia allows establishing clones with low genetic variability and reproductive results (Martinez et al., 1998, as cited in Ramirez y Mendoza 2008). Some authors prefer other species of Daphnia, different from *Daphnia magna* because they have bigger neonates and some fish cannot use them for food (Prieto, 2001).

The Cladocerans species mentioned in this essay have been isolated from the Antioquia State (Ocampo et al., 2010). According to Martinez et al., (1998) the organisms used to do this evaluation must be obtained from laboratories with controlled strains. They can be obtained by purchasing them from distributors of the scientific material; this will guarantee specific and good quality biological material. It is possible to obtain them using recollection although in this case they will have to develop the harvest to an F2, also they must acquire an identity certificate issued by an accredited institution, in this institution they will have to deposit a fixed or live sample, this way it will get a catalog or collection code. It is not allowed to use organisms purchased as feedstuff on a pet store, because the origin, history and quality is unknown. Frequently this material does not guarantee reproducibility and this make the results less reliable (Martinez et al., 1998, as cited in Ramirez y Mendoza 2008, as cited in Ramirez y Mendoza 2008).
