*2.2. Water Physicochemical Analysis, Heavy Metals, and Cyanide Detection*

Some physicochemical parameters were analyzed such as pH (pH/T tester pHep®4, Hanna Instruments, RI, USA) [31], chemical oxygen demand (COD photometer Hanna Instruments, New England, RI, USA) [32], and total solids by the gravimetric method [33]. The detection of heavy metals was performed using a Varian SpectrAA 220 G Atomic Absorption Spectrometer(Varian-Agilent

Inst., Palo Alto, San Francisco, CA, USA), following previous publications: cadmium, chromium, zinc, and nickel [34]; mercury in a direct mercury analyzer (DMA-80, Milestone Inc., Sorisole, Italy) [35]; and cyanide in a portable photometer (Hanna Instruments, RI, USA) [36]. All reactive, analytical standards and reference materials were purchased from Merck (Merck KGaA, Darmstadt, Germany). The results obtained in the samples from Village Gato, Village Tigui, and the water catchment of the Boque River were compared with normative 0631/2015 [37], which establishes the parameters to be monitored and maximum permissible limits in the specific discharges of non-domestic wastewater (precious minerals and gold). While for houses and deep-well underground sites, they were evaluated in compliance with normative 2115/2007 [38], which regulates water for human consumption.

#### *2.3. Bioassays*

In the bioassays, *Lactuca sativa (L. sativa)* [39] and *Hydra attenuata (H. attenuata)* [40] were used as a biological indicators of water quality. After the follow-up of the results of the two first collections of water samples, a modification of the protocol was performed replacing *Daphnia magna (D. magna)* [41] instead of *H. attenuata* due to no evidence of sensitivity against the possible harmful substances that might be present in the water samples by *H. attenuata*. The effects on organisms can be inhibition, sublethality, and lethality volume/volume (v/v). The water samples were diluted in four different concentrations 25%, 50%, 75%, and 100% (v/v); reconstituted hard water (160–180 mg/L CaCO3) was used as diluent for the *D. magna* and *H. attenuata*; while for *L. sativa*, distilled water was used. The response of *H. attenuata* was read using a binocular stereoscope (Leica). Before taking the readings, the containers were shaken in a circular way to reactivate the movement of *D. magna* and confirm their state. In the case of *L. sativa*, graph paper was used to measure the length of the radicle.

#### 2.3.1. Endpoint and Toxic Response Model

*L. sativa*, half-maximal inhibitory concentration (IC50): root growth reduction or inhibitory effects on lettuce seed germination and root growth after 5 days. *D. magna*, lethal concentration (LC50): number dead/total number or lethal effects of water were observed after 48 h of exposure, and *H. attenuata*, median effective concentration (EC50): density reduction or lethality test, produced by irreversible morphological changes after 96 h of exposure.
