2.7.1. Mosquito Rearing

*Culex pipiens* L. mosquitos' vectors were reared in the Laboratory of Medical Entomology, Animal House, Department of Zoology and Entomology, Faculty of Science, Al-Azhar University, Cairo, Egypt. The mosquitoes were preserved at 30–35 ◦C and 60–80% relative humidity with a photoperiod of 12:12 h light: dark. Larvae were reared in white plastic cups (30 cm × 35 cm × 5 cm and containing 500 mL tap water). Fish was added to each tray for 2 weeks for optimum feeding of the larvae as follows: 0.1 g for 1st and 2nd instar larvae, 0.3 g for 3rd instar larvae, and 0.5 g for 4th instar larvae per day, until the pupation stage appeared. New pupae were transferred from the trays to plastic cups containing water and placed in screened cages (size 30 cm × 30 cm × 30 cm) until they emerged as adults. The adults were constantly provided with 5% sucrose solution on saturated cotton pads. The adult female mosquitoes of 5 days were destitute of sugar for 12 h, then fed on human blood by artificial membrane feeding technique [37] for 30 min. After two days of blood-feeding, plastic cups full of tap water were placed inside the cage for oviposition, which occurred on day 3 or 4 after blood feeding.

### 2.7.2. Larvicidal Activity

The larvicidal activity (at room temperature) was assessed by the standard method of the World Health Organization (WHO) with slight modifications using the method described by Velayutham and Ramanibai. [38]. Twenty-five 3rd instar larvae of *C. pipiens* were moved separately from their colony in the laboratory to a plastic beaker (250 mL) containing 100 mL of the biogenic MgO-NPs (10 µg mL−<sup>1</sup> ). The control was set up using chlorinated tap water. The same experiment was repeated with different concentrations of MgO-NPs (8, 6, 4, and 2 µg mL–1). The larval mortality percentages were counted every 24 h for 72 h using the following equation [39]:


The lethal concentrations (LC50) and higher lethal concentrations (LC90) values were calculated at their 95% confidence intervals, as previously reported [40].

#### 2.7.3. Repellent Activity

The repellent study was conducted according to the method described by WHO [41]. This test was monitored by the Faculty of Science, Zoology and Entomology Department, Al-Azhar University, Cairo, Egypt, according to WHO ethics. Briefly, 3-day-old, bloodstarved female *C. pipiens* were held in a net cage (45 cm × 30 cm × 45 cm). The volunteer had no contact with perfumes, lotions, perfumed soaps, or oils for one day before the experiment. From each volunteer arm, only 25 cm<sup>2</sup> of skin, upper side, were uncovered and

exposed to female *C. pipiens*, while the residual arm area was covered with elastic gloves. The biosynthesized MgO-NPs were applied at 1.0, 2.5, 5.0, and 10.0 mg/cm<sup>2</sup> separately to the uncovered arm area. The commercial DEET (N, N-Diethyl-meta-toluamide) was used as a positive control. The repellent experiment was achieved during the night from 7:00 p.m. to 3:00 a.m. The volunteer introduces their arm (control and treated) simultaneously into the cage, gently tapping the sides of the experimental cages to activate the mosquitoes. The experiment was repeated three times for each concentration used. The volunteer was asked to insert their arm (control and treated) into the experiment cage simultaneously for 1 minute every 5 minutes. The mosquitoes that settled on the arm were recorded and then shaken off before sucking any blood. The percentages of repellency were calculated according to the following equation [42]:

$$\text{Repellency percentage}(\%) = \frac{\text{T}\_{\text{a}} - \text{T}\_{\text{b}}}{\text{T}\_{\text{a}}} \times 100\tag{5}$$

where T<sup>a</sup> and T<sup>b</sup> denote the number of mosquitoes in the control and treated groups, respectively.

#### *2.8. Bio-Adsorption and Treatment of Tannery Effluent*

The decolorization of tanning effluents using biosynthesized MgO-NPs was investigated at different concentrations (50, 75, and 100 mg/100 mL) for different contact times (60, 120, 180, and 240 min). The experiment was conducted on a 250 mL conical flask containing 100 mL of tanning effluent mixed with specific NP concentrations. The mixture was stirred for 30 min before the experiment to reach absorption/desorption equilibrium. At the end of each incubation time, 1.0 mL of the mixture (tanning effluent with NPs) was withdrawn and centrifuged at 10,000 rpm for 3 min, and its optical density was measured at the maximum absorption band (λmax) of tanning effluent (550 nm) by a spectrophotometer (721 spectrophotometers, M-ETCAL). The decolorization percentages (%) of tanning effluents were calculated according to the following equation [10]:

$$\text{Decolorization percentage} \text{(\%)} = \frac{\text{C}\_0 - \text{C}\_t}{\text{C}\_0} \times 100\tag{6}$$

where C<sup>0</sup> is the absorbance at zero time and C<sup>t</sup> is the absorbance after specific time t (min).

Based on the most suitable MgO-NPs concentration at the optimum contact time, the biological oxygen demand (BOD), chemical oxygen demand (COD), total dissolved solids (TDS), total suspended solids (TSS), and conductivity were assessed according to the standard recommended methods [43].

The major common tanning heavy metal represented by Cr was measured before and after MgO-NPs treatment using atomic adsorption spectroscopy (A PerkinElmer Analyst 800 atomic spectrometer). The Cr heavy metal was detected by atomic absorption spectroscopy according to the absorption of light by free metallic ions.
