*3.5. Microbiological Analysis*

Table 4 shows the results of the concentration of fecal contamination indicators (total coliforms, *E. coli,* and somatic coliphages) for the different types of water of the five sampling stations. Table 4 shows that concentrations between 103 and 10<sup>5</sup> colony forming unit (CFU)/100 mL for total coliforms were obtained at the different stations. While in the case of *E. coli*, concentrations between 103 and 104 CFU/100 mL were obtained. Somatic coliphages were detected in samples taken at Village Gato and house stations. Colombia does not have regulations for the presence of this indicator, although this is necessary since the presence of somatic coliphages represents a risk to the health of the community.

While, for the water catchment of the Boque River, Village Tigui, and deep-well underground stations, the presence of phages in some samples was not detected (<1.0 <sup>×</sup> 103). The results were compared with decree 1594/1984 [49], which establishes the concentration of total coliforms (2.0 <sup>×</sup> <sup>10</sup>4/100 mL) allowed in waters that will be treated by conventional systems: while normative 2115/2007 [38], establishes that the concentrations for total coliforms and *E. coli* for drinking water is 0 CFU/100 mL.


**Table 4.** Results of total coliforms, *Escherichia coli*, and somatic coliphages in waters of the Boque River and drinking waters.

CFU/100 mL: colony forming units in 100 mL of analyzed water; PFU/100 mL: plaque forming units in 100 mL of analyzed water; n: number of samples analyzed. S: sampling, the numbers 1S, 2S, and 3S correspond to the month of July, September, and December in which the sample was taken; <: less than the limit of quantification; n: is the number of samples.

#### **4. Discussion**

#### *4.1. Bioassays*

#### 4.1.1. *Hydra attenuata* and *Daphnia magna*

By applying the *H. attenuata* toxicity test, it was not possible to determine lethality or sub-lethality since there were no morphological changes in the three samples taken. An important factor that could influence why *H. attenuata* was not sensitive to contaminants present in this water, is that the toxicity of metals is modified by abiotic factors such as hardness, pH, and water temperature [50]. For example, if water hardness is high, the formation of metal complexes tends to increase, which in turn lowers the effect of toxic divalent metals [50,51].

*H. attenuata* has a higher sensitivity to toxic substances at acidic pH, compared to that at alkaline or neutral pH [52]. The pH value of the water sample from the Boque River is about 7 (Appendix A—Tables A1 and A2), which could influence *H. attenuata* not presenting sensitivity when heavy metals, cyanides, or other toxic substances are in the water.

Due to the results obtained with *H. attenuata* in the first two samples, *D. magna* was used in the last sampling, to find an animal indicator that presented a greater sensitivity to these types of contaminants. Table 2 shows different mortality percentages that were found, demonstrating the sensitivity of this organism to the contaminants present in the water of the Boque River that is consumed by the Monterrey population. Studies conducted by Forget et al. [53] with *D. magna*, show percentages of toxicity up to 70% against heavy metals. Castro-Català et al. [54] evaluated the toxicity of sediments and water in rivers with the presence of pesticides and heavy metals using *D. magna* as an animal indicator, showing that it can be sensitive to these types of samples, due to its high metabolic rate [55]. Likewise, Lattuada et al. [56], in southern Brazil, used *D. magna* as an indicator of toxicity in waters affected by coal mining, in which heavy metals such as Fe, Mn, Zn, Ni, Cd, and Pb were found. The results showed sensitivity by this indicator in this type of water and suggest the evaluation of toxicity in waters from gold mining. Moreover, studies conducted in China by Wu et al. [57] demonstrated that the most frequently encountered heavy metals in a region affected by gold mining were mercury and cadmium, as observed in the results found in the drinking water of the population of Monterrey (Appendix A—Tables A1 and A2).

#### 4.1.2. *Lactuca Sativa*

The differences observed between chemical parameters and toxicity may be related to the fact that the samples were not collected simultaneously and that it is not the same water because along the river route and on the different sampling days, diverse factors can alter its quality. Likewise, dilution effects due to rain, sedimentation, the introduction of new pollutants, among others, can have an influence. Additionally, the water entering the treatment plant can be more contaminated, taking into account that it travels through tanks that are not in operation or comes into contact with sludge that might have a higher concentration of contaminants, which may return to the column of water.

The increase in germination, compared to the positive control (overgrowth), is related to the presence of organic matter because they are essential nutrients for *L. sativa* seed germination, and if they are available in high concentrations, they will stimulate growth. On the other hand, mercury and cyanide at the Village Tigui station (Appendix A—Tables A1 and A2) had higher concentrations. The inhibition rates of 24% and 44% for samples 1 and 2 at the concentration of 25%, affect the growth of the seed as the higher concentration of pollutants results in greater inhibition. Castillo et al. [24] found inhibition in the germination of seeds in waters contaminated with mercury and argue that it can occur due to the harmful effects caused by mercury at the cellular level in the seed. These results coincide with other studies where *L. sativa* has been proposed as a useful tool to evaluate and compare the toxicity of industrial effluents that present heavy metal contamination [58].

Likewise, the level of cadmium (Appendix A—Tables A1 and A2), also exceeded the minimum values established by the regulations for water for human consumption; studies have been reported where the exposure of *L. sativa* to this metal causes toxic and harmful effects that decrease its growth as the concentration of Cd, and thereby its adsorption, increases [59–61]. Just as the presence of heavy metals and cyanide has toxic implications in the plant and animal model, in the same way, it will affect the health of the human being [62,63].

Cd is one of the most toxic elements to which man is exposed since the accumulation of this metal in the body is gradual and increases with age due to its long half-life, greater than 20 years [64]. This is why eating food or drinking water with very high levels of cadmium causes severe stomach irritation, which causes vomiting, diarrhea, and sometimes death [64]. Moreover, cyanide exceeded the allowed limits (0.05 mg/L) in one of the samples analyzed in one of the houses (1.11 mg/L). The guide values of the World Health Organization [65] establish that the concentration of cyanide toxic to humans is 0.07 mg/L. Exposure to this concentration or higher may cause inhibition of cell growth, thereby affecting the breathing process and the metabolism of nitrogen and phosphate. It also inhibits the activity of some metalloproteins, joining cofactors such as the heme group of hemoglobin [66].

Finally, cyanide has acute effects on human health such as irritation of the eyes, nose, and throat. High exposure causes intoxication with headache, weakness, nausea, strong heartbeat, coma, and even death. As for chronic effects, it causes nosebleeds and nose lesions and can cause enlargement of the thyroid gland, which can interfere with its regular function [67].

## *4.2. Ames Test*

In some cases, there was a decrease in reverts with increasing doses, which may be due to the presence of toxic substances that prevent the growth of bacteria [68]. However, in most of the sampling stations, a direct relationship was observed between the number of revertants and the increase in the concentration of heavy metals. This demonstrates the high probability of the presence of substances such as heavy metals and organic compounds in the Boque River that cause base-pair mutations and changes in the DNA reading frame of bacteria.

When observing the reversion of the strains, it was evidenced that they exceed 2–40 times the value of the negative control for the TA98 strain in the second sample in one of the houses, and from 2.0 to 2.5 for the TA100 strain in the third sample for the house and underground well. According to Orozco and Zuleta [69], some samples can exceed 100 times the negative control and these results are related to the quality of the water.

Likewise, Meléndez et al. [70] investigated the mutagenic activity of drinking water before and after chlorination at the Villa Hermosa plant, Medellín, Colombia, finding that contamination and chlorination influence mutagenicity. They used the Ames test with strains TA100 and TA98. Sierra et al. [71] evaluated the mutagenic activity of the Cauca River water with the same strains with and without the enzyme activator S9, finding that the highest rate of mutagenicity was observed with

strain TA98 without enzyme activator. However, the TA100 strain is characterized by presenting the *hisG46* mutation and has specific markers that give it greater sensitivity to the test; within these are the *uvr* mutation, the *uvrB* mutation, and the plasmid pKM101 [26,68].

Mesquidaz et al. [72] reported alarming figures in the mercury concentrations used in the gold extraction process in a mine in northern Colombia, which ranges from 50 to 100 tons in 2007. Furthermore, it is reported that, thanks to this pollutant present in water, the health of the population has been affected, since Hg was found in human hair at a concentration of 12.8 μg/g, a figure that is well above international standards. It has been shown that inhabitants of different municipalities in southern Bolívar where gold mining takes place have high levels of Hg contamination, and this situation requires special attention to reduce environmental and human health problems [4]. Mercury contamination has been linked to health problems, as direct absorption of mercury vapor released by incinerators in gold mining, or ingestion of mercury-containing wastes, causes hydrargyrism and poisoning. Mercury (Hg) is one of the heavy metals of greatest concern to populations that consume fish. This pollutant can be released from many sources and has various toxic effects in humans [73].

Some of the health problems caused are excessive salivation, shortness of breath and fatigue, bronchitis, tremors and irritability, personality changes (due to brain damage), a sensation of floating teeth and pain in them, kidney and respiratory damage that can lead to death from problems in the lungs and other organs of the body [74–76]. While breathing polluted air, elemental mercury can reach the brain, affecting nerve cells and the olfactory system. The main organs in which mercury accumulates are the brain and the kidney [77,78].
