*2.4.3. BMWP scores for macroinvertebrates families*

To assign bioindication values to the different macroinvertebrates families, a data matrix of sampling sites *vs* abundance was constructed using the mean abundance values for each aquatic macroinvertebrate family from all sampling and study sites. The mean abundances were standardized to six abundance classes: class 0 (0 organisms), class 1 (1–3 organisms), class 2 (4–10 organisms), class 3 (11–33 organisms), class 4 (34–100 organisms) and class 5 (>100 organisms). This standardization was carried on following [14] to reduce the possible effect of the overvaluation by the local dominance of some groups due to the nonhomogeneous natural distribution of the macroinvertebrates.

For each family of macroinvertebrates, the abundance class data were pooled within each *Pcq* interval. In cases where a family appeared in more than one study site within the same *Pcq* interval, the abundance classes of such sites were averaged in order to obtain a single abun‐ dance value per family for each *Pcq* interval. The value of class abundance obtained by a *Pcq* interval indicates the number of times you have to replicate the value of the superior limit corresponding to the *Pcq* interval; for example, if a family obtained a value of 1 for its abun‐ dance class within the 0–1 *Pcq* interval, a 3 for the 3–4 *Pcq* interval, a 4 for the 5–6 *Pcq* interval and a 2 for the 6–7 *Pcq* interval, the data for calculating the fifth percentile would be 1, 4, 4, 4, 6, 6, 6, 6, 7 and 7. The bioindication values for each aquatic macroinvertebrate family were calculated by obtaining the fifth percentile of the abundance class distributions along the *Pcq* intervals where that family was present. This value represents the minimum tolerance value of this family in relation to organic pollution; in the case of this example, the fifth percentile would be 2, which is the family BMWP bioindication value.

### **2.5. Definition of BMWP water quality categories**

The water quality category ranges for the BMWP values were assigned following [21]. The median value of the data set of the reference sites was calculated. Scores above this median value will correspond to the "Excellent" quality category, while values that fall between the median and the tenth percentile of that distribution are considered to be in the "Good, not sensible affected" quality category. Values below the tenth percentile were subdivided into four equal parts, which correspond to the categories "Regular,""Bad, polluted,""Bad, very polluted," and "Bad, extremely polluted." The names assigned to each of the water quality categories with some modifications were those proposed by Alba‐Tercedor [29]. The selection of reference conditions included physical, chemical and biological criteria (WQI, *Pcq*, land use and macroinvertebrate community) following [30].

### **2.6. BMWP statistical validation**

The validation process was performed using three approaches. First, a score prediction test, proposed by Armitage et al. [31], with the average per study site of BMWPobserved *vs* BMWPexpected values. The expected values were calculated with a multiple linear regression (best model procedure) with the qualifying variables for each study site. The observed and expected values were plotted and confidence intervals (*α* = 0.05) were calculated using the XLSTAT software 2013. The goodness of fit of the model was evaluated using the coefficient of determination (*R*<sup>2</sup> ) and *p* values.

For the second validation approach, the degree of fit of the model for the BMWPobserved *vs* BMWPexpected was tested using two independent methods: the [32] efficiency model (NSE) (range −∞ to 1.0), a widely used statistic for hydrological models

A third approach for the index validation and for the assessment of the geographical extension of the BMWP calibrated was performed with additional information that was obtained from the National Agency for Water in México (CONAGUA), data included aquatic macroinverte‐ brates collected in the county of Morelos. For the index validation, nine sites within the Apatlaco subbasin were considered: Arriba Chalchihuapan, Arroyo Chapultepec, water treatment plant (WTP) Acapatzingo, WTP Emiliano Zapata, WTP El Rayo, Apatlaco‐Xochite‐ pec, WTP Xochitepec, WTP Zacatepec and Tlaltenchi (**Figure 1b**). For the geographical extension, data from CONAGUA of seven sites in three subbasins were considered: Amacuzac subbasin, sites Chontalcoatlán, Amacuzac and Arroyo Salado; Cuautla subbasin, sites Barran‐ ca Santa María and Papayos; and Yautepec subbasin, sites Pedro Amaro and WTP Jojutla. These sites belong to the Balsas Basin and the last three subbasins are adjacent to the two rivers monitored in this study (**Figure 1b**). The monitoring team of CONAGUA used D‐nets (mesh size of 500 µm), a multihabitat sampling and each habitat was sampled in 1 m2 , with three replicates. Based on the calibrated scores for aquatic macroinvertebrates, the BMWP scores were calculated for each site of the data set from CONAGUA and these scores were included in the previous model generated with *observed vs expected* data for the BMWP with confidence limits (*α* = 0.05). A Pearson correlation analysis was also performed for the BMWP.
