**2. Materials and methods**

#### **2.1 Study design**

This experimental study was performed in the Laboratory of Photochemistry and Photobiology and in the Environmental Laboratory, at the University of the Region of Joinville. It involved the *Euglena gracilis* KLEBS algae from the University of Göttingen's collection, Germany. Behavioral changes, photosynthetic activity and chlorophyll level alterations were analyzed, when the algae were submitted to chlorinated water from the pharmacy, as well as the raw effluents and the post-ozone/UV-POA effluents.

#### **2.2 Sample collection**

Three types of samples were analyzed:


All of them were collected from the four pharmacy laboratories.

In order to conduct the study, there was the collaboration of a magistral pharmacy from Joinville city, northeast of Santa Catarina, the same facility that was part of Pinto et al.'s investigation [8]. The pharmacy made possible the samples collection from the four production environments of the pharmacy:

• psychotropics laboratory, responsible for the manipulation of controlled-sale medicine prescriptions, accordingly to the Order 344/98;


The samples considered as water of access were collected directly from the faucets of the washing sinks in each laboratory, through previously sterilized borosilicate glass jars. They were used here as control samples (water from the wastewater treatment plant). The raw effluents samples were collected from the syphons connected to the washing sinks using a peristaltic pump, and here sterilized borosilicate glass jars were also used. Samples were taken up to 12 L−1 from each laboratory. Afterwards, the samples were kept in polystyrene boxes with ice and away from light up to their packaging in the freezer.

#### **2.3 Production estimate of the magistral pharmacy laboratories**

In order to conduct the study, there was the collaboration of a magistral pharmacy from Joinville city, northeast of Santa Catarina, that made possible the samples collection from the four production environments of the pharmacy.

An important factor to be considered here is the quantity of actives and other substances disposed through the sinks and that compound the laboratories' raw effluent. For this purpose, the pharmacy's average monthly production of six months was taken into account, and the monthly average production was calculated, in order to verify the laboratories' activity average, accordingly Eq. (1):

$$MP = \left(\frac{\sum \text{Monthlyquantity of products}}{30}\right) \times \left(\frac{\sum \text{mg} of request made}}{\text{quantity of requests}}\right) \tag{1}$$

#### **2.4 Fractions tests preparation**

#### *2.4.1 Removal process*

Removal reaction occurred in a 500-mL−1 reactor, which contained the raw effluent samples from all the studied laboratories. The other samples removals occurred after 1-hour ozonation, through a Trump TCB ozone generator, that injected the ozone in a 10-mg L−1 flow.

The total time was of 2 hours, in accordance with Ferreira [9]. The volume removed was up to 10% of the total volume (50 mL), following the recommendation, to avoid interferences related to a larger oxidizing agents' exposure to a smaller contaminant volume [12]. Afterwards, the samples were kept in freezer to be later analyzed.

#### **2.5 Tests for environmental toxicity risks**

For the purpose of tests for environmental toxicity risks, four sample categories were investigated:

*Evaluation of the Use of Advanced Ozone Oxidative Process in Reducing the Danger… DOI: http://dx.doi.org/10.5772/intechopen.95068*


## *2.5.1 Tests with Euglena gracilis algae*

From each one of the four sample categories, a 5-mL aliquot was removed, and it was added in a 40-mL *Euglena gracilis* algae culture. The collections were performed for photosynthetic efficiency tests, chlorophyll concentration and behavioral evaluation via NGTOX after a period of at least 48 hours, according to Ekelund [13].

#### *2.5.1.1 Algae photosynthetic efficiency test via PAM*

For testing the photosynthetic efficiency via PAM, the photosynthetic parameters were measured through a modulated pulse-width PAM 2000 fluorimeter (Walz, Effeltrich, Germany). The PAM measurement principle is based on changes on the chlorophyll fluorescence level, after the application of saturated light pulses. The photosynthesis performance (Yeld) was, then, calculated accordingly Eq. (2), on Yeld photosynthetic efficiency.

$$Yield = \frac{fm - f\mathbf{0}}{f\mathbf{0}}\tag{2}$$

Approximately 5 mL−1 of the tested cultures were taken and transferred to the cuvette of the PAM equipment. They were then submitted to saturating light pulse emission, for photosynthetic activity evaluation. The saturating light pulse emission made possible the detection of the maximum fluorescence Fm, indicating total reduction of the electrons FSII receptor. The light-curve response was determined for all the treated samples. The algae were exposed to an increasing luminous intensity (generated by an internal halogen bulb) in 10 steps, from 0 to 3111 molm-2/s. After 10 s of each luminous step, a saturating pulse was applied, and the photosynthetic performance and the electron transport rate were measured automatically.

Afterwards, the average performance on test situation was calculated, considering all the values obtained on the saturation process. The global photosynthetic efficiency was measured according with Eq. (2):

$$EFG = \frac{\sum \text{Yield during saturation}}{\text{Quantity of submitted irradiating pulses}} \times 100\tag{3}$$

This way, it was intended to analyze the interference the compound of raw effluents and the waste will promote in the algae culture, when compared to the control one.

#### *2.5.1.2 Evaluation of algae behavioral changes through biomonitoring via NGTOX*

The behavioral tests with *Euglena gracilis* and the samples from the laboratories were conducted using a real-time biomonitoring tool called NGTOX, developed

and homologated by Ecobabitonga Tecnologia Ltda. The instrument has monitored, through the analysis of real-time images, the algae behavior, considering different movement parameters of the photosynthetic single-celled flagellate [12].

The equipment is a system of connections that involve four silicon tubes responsible for the suction of *Euglena gracilis* cells culture, water samples with hormones for the test, water for sample dilution and the analyzed material disposal.

Three pumps activated by peristaltic motors transported the cells, the diluent and the sample up to a glass cuvette of 22 mm of internal diameter and 0.2 mm of thickness. The trial bodies in contact with the control were blended and transferred to an observation cuvette, connected to a microscope, which captured the images of the cells in movement. The images were recorded by a charged coupled device camera and digitalized by a board connected to a microcomputer, in which they were presented in a monitor. Then, the software calculated the movement parameters, the movement speed, the ascent rate, the average cell size, etc. After that, the samples were added separately, and the analyses were performed by the software one more time 10 minutes later. Any alteration on the movements, average speeds, ascent rates and cell size were calculated and compared with the previous results [14].

### *2.5.1.3 Test for alterations on concentration of chlorophyll present in algae: chlorophyll removal from the algae and UV analysis (160 SHIMADZU)*

This test had the objective of verifying if the parameters previously analyzed affected the chlorophyll concentration. After the time of exposure to contaminants, 5-mL of the culture media submitted to the presence of the test samples and the control were taken. These aliquots were treated according with the procedures conducted by [15]. Aliquots were vacuum-filtered through Whatman® 47-mm filter paper.

The papers containing the filtered (precipitated cells) were transferred to a Falcon tube, received 5 mL of ethanol and were afterwards kept at 4°C for 60 minutes, for the pigments extraction. Then, the mixtures were centrifugated at 6000 g for 10 min at 4°C in order to aggregate on the waste cells.

The absorption spectrum of the supernatant was measured accordingly Lorenz's equation for the calculation of chlorophyll concentration.

#### **2.6 Data statistical analysis**

The data were evaluated through ANOVA, a univariate technique which deals with the quantitative data relating them to three-level independent category variables.

For the groups' analysis (tests and control), comparing all the effects, the used technique was an ANOVA extension, called ANOVA for repeated measures, that consists on a better developed approach for paired data. Then, comparisons of results and averages based on the samples' quantitative items were performed.

Following, the other variables were described, since formally there is not a statistical hypothesis test, although it works on confirming or not the a-priori expectations about the results.

The statistical analysis on algae behavior evaluated via NGOTX were conducted by ImagingTox®, a software especially developed and written for Microsoft platform, with multilingual Net 64-bit and MS SQL Server database. It has seven threads (the main one, three for video 1 and three for video 2), two functions (one for controlling the PC and NGTOX connection and the other one for database connection and validation), making possible the storage of bioassays performed for forensic analysis and real-time results exhibition screen. ImagingTox® conducted the 5-PL integrated statistical analysis.

*Evaluation of the Use of Advanced Ozone Oxidative Process in Reducing the Danger… DOI: http://dx.doi.org/10.5772/intechopen.95068*
