# Anthropogenic eutrophication of Lake Titicaca (Bolivia) revealed by carbon and nitrogen stable isotopes fingerprinting

C. Heredia, S. Guédron, D. Point, V. Perrot, S. Campillo, C. Verin, M.E. Espinoza, P. Fernandez, C. Duwig, D. Achá

► To cite this version:

C. Heredia, S. Guédron, D. Point, V. Perrot, S. Campillo, et al., Anthropogenic eutrophication of Lake Titicaca (Bolivia) revealed by carbon and nitrogen stable isotopes fingerprinting. Science of the Total Environment, 2022, 845, pp.157286. 10.1016/j.scitotenv.2022.157286. hal-04813911

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Anthropogenic eutrophication of Lake Titicaca (Bolivia)
revealed by carbon and nitrogen stable isotopes fingerprinting

C. Heredia<sup>a,b*</sup>, S. Guédron<sup>a,c</sup>, D. Point<sup>b,d</sup>, V. Perrot<sup>a</sup>, S. Campillo<sup>a</sup>, C. Verin<sup>a</sup>, M.E. Espinoza<sup>b</sup>, P. Fernandez<sup>b</sup>, C. Duwig<sup>c,e</sup>, and D. Achá<sup>b</sup>

<sup>a</sup>Université Grenoble Alpes, Université Savoie Mont Blanc, CNRS, IRD, IFSTAR, ISTerre,
38000, Grenoble, France.

<sup>b</sup>Instituto de Ecología, Unidad de Calidad Ambiental (UCA), Carrera de Biología, Universidad Mayor de San Andrés, Campus Universitario de Cota Cota, casilla 3161, La Paz, Bolivia.

<sup>c</sup>Laboratorio de Hidroquímica - Instituto de Investigaciones Químicas - Universidad Mayor de San Andrés, Campus Universitario de Cota-Cota, casilla 3161, La Paz, Bolivia.

<sup>d</sup>Géosciences Environnement Toulouse (GET) - Institut de Recherche pour le Développement (IRD), CNRS, Université de Toulouse.

<sup>e</sup>Univ. Grenoble Alpes, Univ. Savoie Mont Blanc, CNRS, IRD, IFSTAR, IGE, 38000 Grenoble, France.

Carlos Heredia A.; carlos.heredia-aguilar@univ-grenoble-alpes.fr

<footnote>\*Corresponding author:</footnote>
## Highlights

* C and N stable isotopes were measured in four compartments of Lake Titicaca

* Anthropogenic discharge affects baseline C and N isotope signatures

* C recycling from soil, anthropogenic and lacustrine sources affect the δ¹³C signature

* δ¹⁵N signatures track anthropogenic contamination independently of plant type

* Decreased anthropogenic contribution in the bay assessed by isotope mixing model
Graphical abstract

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Abstract

Cultural eutrophication is the leading cause of water quality degradation worldwide. The traditional monitoring of eutrophication is time-consuming and not integrative in space and time. Here, we examined the use of carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopic composition to track the degree of eutrophication in a bay of Lake Titicaca impacted by anthropogenic (urban, industrial and agricultural wastewater) discharges. Our results show increasing δ¹³C and decreasing δ¹⁵N signatures in macrophytes and suspended particulate matter with distance to the wastewater source. In contrast to δ¹⁵N and δ¹³C signatures, in-between aquatic plants distributed along the slope were not only affected by anthropogenic discharges but also by the pathway of carbon uptake, i.e., atmospheric (emerged) vs aquatic (submerged). A binary mixing model elaborated from pristine and anthropogenic isotope end-members allowed the assessment of anthropogenically derived C and N incorporation in macrophytes with distance to the source. Higher anthropogenic contribution was observed during the wet season, attributed to enhanced wastewater discharges and leaching of agricultural areas. For both seasons, eutrophication was however found naturally attenuated within 6 to 8 kilometers from the wastewater source. Here, we confirm that carbon and nitrogen stable isotopes are simple, integrative and time-saving tools to evaluate the degree of eutrophication (seasonally or annually) in anthropogenically impacted aquatic ecosystems.

**Keywords:** carbon stable isotopes, nitrogen stable isotopes, eutrophication, Lake Titicaca, anthropogenic discharges
1. **1. Introduction**

Human-induced eutrophication of surface waters has become a major concern of water quality impairment (Smith, 2003; Schindler, 2006). It is known to impact aquatic ecosystems (Backer and McGillicuddy Jr, 2006; Paerl and Otten, 2013) with environmental and health-related costs estimated to several billions of dollars per year (Pretty et al., 2003; Dodds et al., 2009). The discharge of untreated wastewater in natural aquatic ecosystems induces a nutrient enrichment in the water column, affecting the trophic structure and composition of the in biological communities (Smith et al., 2006). A common response to the excess of nutrient inputs is the possible development of massive algal proliferation (i.e., blooms) which provokes oxygen depletion (i.e., hypoxia or anoxia) during the bacterial degradation of senescent blooms that can result in the death of benthic and pelagic biota (Diaz, 2001). Recurrence of such hypoxic events can cause the release of phosphorus from sediments (due to changing redox conditions), creating an internal feedback that amplifies eutrophication effects (Rosenmeier et al., 2004; Vahtera et al., 2007; Smith et al., 2015).

The monitoring of eutrophication generally includes cost and time-consuming *in situ* physicochemical analyses (pH, redox potential, total nutrient concentration, biochemical oxygen demand, etc...) as well as macrophyte and algal species composition and biomass (Le Moal et al., 2018). In impermanently eutrophic ecosystems or those subject to occasional blooms, such traditional monitoring methods require a large number of measurements to obtain a significant spatial overview of the anthropogenic impacts, which are generally not constrained temporally. Therefore, the need for a different approach with sensitive techniques, providing a spatially and temporally integrative picture of eutrophication processes, is of high priority to understand the dissemination and triggering mechanisms of blooms. This latter
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information is key for the development of mitigation strategies before irreversible changes
affect aquatic ecosystems.

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Carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope are good candidates to provide such level of information at the ecosystem scale (Fry, 2006; Dawson and Siegwolf, 2007; Glibert et al., 2018). Both C and N isotopic tracers provide information about the food web structures and are influenced by baseline changes and sources (Vander Zanden and Rasmussen, 1999; Liu et al., 2012). δ¹³C allows the inference of energy sources (i.e., no significant fractionation of C isotopes during trophic transfer, resulting in isotopic ratios between consumers and their food almost identical), whereas δ¹⁵N allows the determination of trophic levels (i.e., enrichment factor between consumers and their food is between 2 and 4 ‰) (Dawson et al., 2002; Michener and Lajtha, 2008; Middelburg, 2014). In addition, when a significant difference in isotopic signatures exists between natural and anthropogenic sources, it is possible to track and assess the magnitude of anthropogenic pollution (Glibert et al., 2018). On the one hand, carbon stable isotope ratios of organic matter (δ¹³C<sub>org</sub>) can be used in freshwater ecosystems to distinguish between autochthonous and allochthonous sources of organic matter (Gu et al., 2006; Thevenon et al., 2012). δ¹³C<sub>org</sub> values of terrestrial primary productivity often differ from the δ¹³C<sub>org</sub> values of aquatic primary productivity (Fry, 2006). On the other hand, nitrogen stable isotope ratios (δ¹⁵N) can track anthropogenic nitrogen inputs (Glibert et al., 2018). The three primary sources of anthropogenically derived N in freshwater ecosystems are human and animal (e.g. septic and manure waste) sewage, synthetic fertilizers (from agricultural fields) and atmospheric deposition (Cole et al., 2006), with each of these sources often differing in their δ¹⁵N signature. Sewage-derived N commonly presents δ¹⁵N values enriched in heavier isotopes [≥10 ‰, ]) relative to the baseline value of atmospheric deposition (N₂), which is closer to 0 (Kendall et al., 2008; Holtgrieve et al., 2011). Once discharged in surface or ground water,

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anthropogenic N recycling (i.e., nitrification or denitrification) can cause large N isotope
fractionation up to ±20 ‰ (Mariotti et al., 1988; Sebilo et al., 2003; Kendall et al., 2007).
Changes in the isotopic composition of dissolved inorganic pools are reflected in the δ¹³Corg
and δ¹⁵N of primary producers, which can assimilate dissolved nutrients from the water
column and integrate the changes in isotopic sources over time (Schindler et al., 1997; Savage,
2005; Dawson and Siegwolf, 2007). Consequently, the δ¹³C and δ¹⁵N values of primary
producers can alert the presence of anthropogenic nutrients before significant ecological
changes occur in the environment (McClelland et al., 1997; Savage, 2005; Vermeulen et al.,
2011). Enriched or depleted isotope ratios in relation to a “pristine” baseline can therefore be
used as sensitive indicators of pollution (Costanzo et al., 2001).

In many developing countries, the demographic expansion and the development of urban
centers and economic activities (e.g., mining, agriculture, industry) increase anthropogenic
impacts on their surrounding ecosystems. This has been the case during the last few decades in
the high-altitude Andean Altiplano (Mazurek, 2012), where the discharge of untreated
wastewater from cities like Puno, Oruro, and La Paz/El Alto have led to the degradation of
several water bodies. The most dramatic examples are the case of Lake Uru-Uru (Bolivia)
(Alanoca et al., 2016; Guédron et al., 2017; Sarret et al., 2019), Puno Bay (Peru) in the northern
basin of Lake Titicaca (Northcote, 1992), and Cohana Bay (Bolivia) in the southern basin of
the Lake (Archundia et al., 2017a; Flores Avilés et al., 2022). Besides the permanent state of
eutrophication in several shallow basins receiving untreated wastewater, punctual and
sometimes massive bloom events have been reported at larger scales. For example, in 2015,
Lake Titicaca suffered one of its major bloom event to date at the scale of its entire southern
basin, triggered by an unusually intense rain episode that washed out large amounts of
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nutrients from the agricultural watershed and resulted in the massive death of birds and pelagic
biota (Achá et al., 2018).

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In this study, we examined δ¹³C and δ¹⁵N signatures in primary producers (macrophytes, periphyton), suspended particulate matter, and surface sediments along an eutrophication gradient in Lake Titicaca. The aim of this study is to test the efficiency of these tools for monitoring the dissemination and amplitude of anthropogenic discharges of wastewaters while considering the natural functioning of the ecosystem (trophic status and seasonality).

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## 2. Material and Methods

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### 2.1. *Study area*

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Lake Titicaca is the largest freshwater lake in South America. Located in the high tropical Andes (3809 m.a.s.l.) between Peru and Bolivia, it is divided into two separated basins: the northern great lake, namely Lago Mayor (7131 km²; mean depth = 100 m; max depth = 285 m), and the southern small lake, namely Lago Menor (1428 km²; mean depth = 10 m; max depth = 40 m) (Fig. 1a). Both basins are connected through the Strait of Tiquina (approx. 800 meters wide). The only outlet of the system, the Desaguadero River, is located in the southern end of Lago Menor (Dejoux and Iltis, 1991). Its high elevation results in extreme hydro-climatic conditions, e.g., intense UV-radiation (Richerson et al., 1986; Villafae et al., 1999), lower dissolved oxygen (Achá et al., 2018), and large daily temperature variations which can overwhelm seasonal ones (Molina et al., 2014).

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The climate of the Titicaca region is marked by a seasonal cycle of precipitation, and more than 70% of the precipitation occurs during the austral summer (mean annual precipitation ~614 mm year⁻¹), concentrated between December and March, when moist air from the

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Amazon enters the Altiplano and discharges in the form of convective rainfalls (Guédron et al., 2018; Segura et al., 2019). Precipitation during the Austral summer produces intense and short duration floods of the lake's tributaries on the steep slopes of the lake (Roche et al., 1992).

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**Figure 1.** Map of Lake Titicaca, presenting the sampling site location of a) control points in the coastal (CH = Chua, HU = Huatajata) and deep open waters (Lm and L3) of Lago Menor; b) the kilometric sampling points performed as a transect in the eutrophicated area of Cohana from the Katari River outlet (CB 1) to the open lake (CB 6) and from the weakly eutrophic Bay of Quehuaya (Q1 and Q2) for comparison and c) ecological repartition of the aquatic vegetation in Cohana Bay.

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This is the case for the Katari River, located in the southwestern part of Lake Titicaca, which exhibits water discharges up to 151 m³.s⁻¹ during flood event, whereas its average annual discharge is 5.8 m³.s⁻¹ (SENAMHI, 2020). The river drains a catchment area of more than 484
105 km², and is supplied by two perennial tributaries, i.e., Rio Seco (slope ~ 0.9%) and Rio Pallina
106 (slope ~ 0.03%) and many ephemeral tributaries (Chudnoff, 2009). In its upstream part, the
107 two perennial tributaries receive both treated and untreated wastewaters incoming from
108 upstream cities [i.e., El Alto City (1.2 million habitants) and Viacha (80,000 habitants)]
109 (Chudnoff, 2009; Archundia et al., 2017). Downstream, the Katari river drains a flat
110 agricultural areas where at least 65 % of the population is dedicated to agricultural and
111 livestock practices (Flores Avilés et al., 2022). Currently, only small amounts of inorganic
112 fertilizers are used in agricultural pratices (Martinez Gonzales et al., 2004; Aguilera et al., 2013).
113 The Katari River flows into the Cohana Bay (Fig. 1b), a large (> 86 km²) and shallow area
114 (average depth ~ 3 m) located in the southwestern part of the southern basin of Lake Titicaca.
115 In the Bay, sediments are covered with totoras (Schoenoplectus californicus) in the inner margin (0-
116 2 m depth) and by macrophytes (mostly Characeae spp.) in the photic zones (i.e., ~ 15 m depth)
117 with maximum development between the depths of 4.5 and 7.5 m (Fig. 1c) (Collot et al., 1983;
118 Dejoux and Iltis, 1991).

119 2.2. *Sampling location and sample conditioning*

120 Sampling was carried out in both April and September 2013, at the end of the wet and dry
121 seasons, respectively. Six sampling sites were chosen within the Cohana Bay, starting from the
122 confluence of the Katari River (KR) in the lake, and continuing towards the interior of the lake
123 (CB 1 to CB 10.6) (Fig. 1c).

124 Sites were chosen following a eutrophication gradient previously determined by surface
125 concentrations of dissolved organic carbon (DOC, Fig. 2). Two sampling zones outside
126 Cohana Bay were chosen for comparison: i) the upper adjacent Bay of Quehuaya (Q1 and Q2)
127 and Huatajata (HU) as representative of coastal sites exhibiting a weak eutrophication, and ii)
128 three remote sites considered as non-impacted by anthropogenic activities: Chua (CH), the
129 center of the open lake (L3) and just downstream of the Strait of Tiquina (Lm) which
130 represents the water of the Lago Mayor (Fig. 1a).

131 All samples were obtained wearing nitrile gloves and collected in 50 ml polypropylene Falcon
132 tubes to avoid cross-contamination. Macrophytes were collected manually or by dredging for
133 the deep submerged ones. Five species belonging to four families were collected:
134 Schoenoplectus californicus (Cyperaceae), Myriophyllum sp. (Haloagaceae), Elodea sp.
135 (Hydrocharitaceae), Chara sp. (Characeae), and Stuckenia sp., Zannichellia sp.
136 (Potamogetonaceae). Periphyton were collected manually by scraping them from the surface of
137 substrate macrophytes. A Van Dorn bottle allowed the collection of water samples for SPM,
138 then sieved and filtrated through pre-combusted glass microfiber filters (Whatman ®, GF/F,
139 47 mm diameter, 0.7 µm mesh). Surface sediments were collected using a gravity corer.

140 Samples of macrophytes, periphyton, sediments (wet season only) and suspended particulate
141 matter (SPM, dry season only) were collected at each site, except for the Chua, L3 and Lm sites
142 where only POM was collected. Samples of superficial soil directly upstream of the Katari
143 River inlet were also collected. All samples were stored at -20 °C and freeze-dried before
144 laboratory analyses.

145 **2.3. Chemical Analysis**

146 Physicochemical parameters (pH, redox potential, conductivity, dissolved oxygen, etc...) of the
147 water column at each site were measured with a HANNA HI-9828 submersible
148 multiparameter probe (Hanna Instruments, Woonsocket, RI, USA).
Major anions concentrations were determined by ion chromatography (Dionex ICS 2000) and hydrogen sulfides by HPLC-UV following published procedures (Achá et al., 2018; Guedron et al., 2020).

Dissolved Organic Carbon (DOC) concentrations were determined by a Non-Dispersive Infra-Red (NDIR) CO₂ Shimadzu® (Model VCSN) spectrometer after humid oxidation in a sodium persulfate solution at 100 °C at the geochemistry-mineralogy platform of ISTerre (Grenoble, France).

Cavity Ring-Down Spectrometry (Picarro, Inc.®) coupled with Combustion Module (Costech, Inc.®) (CM-CRDS) allowed determination of δ¹³Cₜool of SPM as well as δ¹³Corg of macrophytes, soils and sediments at the geochemistry-mineralogy platform of ISTerre of the University Grenoble-Alpes. Samples were calibrated against IAEA-603 standard. Analytical methods, calibration, and sample preparation (including decarbonatation) are detailed elsewhere [Paul et al. (2007), Balslev-Chausen et al. (2013), Cossa et al. (2021)].

δ¹⁵N of macrophytes, periphyton, SPM and sediment were measured using a Delta V Plus continuous flow isotope ratio mass spectrometer (CF-IRMS) (Thermo Scientific, Brême, Germany) with a ConFlo IV universal interface and ZeroBlank autosampler (Costech, Milan, Italy), after a complete combustion of samples on a Flash EA1112 Elemental Analyzer (EA) (Thermo Scientific, Milan, Italy) at the LIENSs laboratory of the University of La Rochelle (France). Samples were calibrated against IAEA-N1, -N2 and -N3 standards.

The analytical precision was ± 0.11 ‰ (n = 27) for nitrogen (N₂) and ± 0.18 ‰ (n = 21) for carbon (CO₂). The isotopic composition was reported in relation to international standards for carbon (Vienna Pee Dee Belemnite - VPDB) and nitrogen (air) respectively. The data were
171 expressed according to the deviation in parts per thousand (‰) from the respective standards,
172 following the notation for isotopic composition (δ).

173 **2.4. Statistical Analysis**

174 Statistical analyses were performed using SigmaPlot version 12.5. *P* values (p) and correlation
175 coefficients (R) are reported for linear regression analyses. In all cases, a *p*-value of 0.05 was
176 chosen to indicate statistical significance.

177 **3. Results and discussions**

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179 **3.1. Natural mitigation of anthropogenic wastewater discharge in the Cohana Bay**

180 Physicochemical parameters along the Cohana Bay towards the open lake are presented in
181 **Figure 2.** The highest values of pH, conductivity (up to 1000 µS cm⁻¹), and total dissolved
182 solids (TDS; up to 500 ppm) are found in the deepest part of the open Lake (i.e., Chua), which
183 is consistent with its reported relatively saline and alkaline waters (Dejoux and Iltis, 1991) that
184 result from the erosion of sulfate-rich minerals from the catchment (Banks et al., 2004; Ramos
185 Ramos et al., 2014). Dissolved oxygen content is also near oxygen saturation at both sites due
186 to the daily mixing of the water column (Dejoux and Iltis, 1991). Similar values observed in
187 the coastal site of Huatajata supports that these sites can be considered a weakly impacted area.

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<figure><img src="image_26.png" /><figcaption>**Figure 2.** Physicochemical parameters in surface water (1.5 meters deep) of Cohana Bay (CB 1 to CB 6), the open lake (CH=Chua) and coastal sites (HU=Huatajata, Q1 and Q2 = Bay of Quehuaya) measured in April 2013. DOC was measured in April (wet season) and September (dry season) 2013. Regressions are plotted only for p values < 0.05. The colored area represents</figcaption></figure>
193 the wastewater input from the Katari River (KR). The three comparative sampling zones are indicated on the top of the figure with
194 gray bands.

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196 In contrast to the open lake, the physicochemical parameters of Cohana Bay's surface waters
197 depict major gradients along the transect, illustrating the change in water quality between the
198 inlet of the Katari river and the open lake. Previous publications (Archundia et al., 2017a;
199 Archundia et al., 2017b) have documented the quality of the Katari River network, showing
200 that water arriving at the lake had high DOC and nutrient contents but low dissolved oxygen
201 and slightly acidic to neutral pH. The physicochemical dataset obtained downstream the
202 confluence of the Katari River (i.e., CB1 and CB 2), confirms the significant contribution of
203 urban (i.e., El Alto City and Viacha City) and agricultural areas in the discharges of DOC to the
204 downstream freshwater ecosystem, a common feature of human-impacted watersheds [Stanley
205 et al. (2012), Aitkenhead-Peterson et al. (2009)]. Higher DOC concentrations were found in
206 this area during the dry season compared to the wet one (e.g. 23.1 and 6.5 mg L⁻¹ at CB 2 for
207 the dry and wet season, respectively, Fig. 2), which illustrates the dilution of DOC in
208 wastewater during the wet season (December to April).

209 High concentration of DOC (and its organic acids) produces water acidification (Kortelainen,
210 1999) and the depletion of dissolved oxygen (Lindell et al., 1995) as observed in the first 5 km
211 downstream the Katari River inlet. Strikingly, DOC concentrations decreased gradually along
212 the transect in parallel with the rise in pH, conductivity, TDS, and dissolved oxygen from the
213 Katari River inlet site (CB 2) to the interior of the bay (CB 6). Hence, this illustrates the
214 attenuation of eutrophication within the Cohana Bay, where the water quality changes from
215 anoxic, highly reducing and moderately acidic in the first kilometers from the inlet (CB 2), to
216 oxic and alkaline but still reducing waters at 7.4 km (CB 3), and finally reaching similar quality status as the open lake (i.e., HU and CH) at 10.7 km (CB 6). In contrast, nitrate ($NO_3^-$) concentrations decreased within the first 5 km (from 5.2 to 0.8 µmol L$^{-1}$) whereas no variation in nitrite ($NO_2^-$, average = 0.13 ± 0.07 µmol L$^{-1}$) nor phosphate ($PO_4^{3-}$, average = 0.18 ± 0.05 µmol L$^{-1}$) were observed along the bay. Based on the annual average Katari river discharge of 5.8 m$^3$.s$^{-1}$ (SENAMHI, 2020), and average nitrate (3.3 ± 2.1 mg.L$^{-1}$) and phosphate (5.6 ± 3.1 mg.L$^{-1}$) concentrations in the lower Katari catchment (Archundia et al., 2017; Sarret et al., 2019); the annual nitrate and phosphate discharge to the bay are 0.6 ± 0.4 Mg.y$^{-1}$ and 1.0 ± 0.6 Mg.y$^{-1}$. The low concentrations in nutrients at CB 2 are consistent with those reported by Achá et al. (2018) for the Cohana area, but at least three times lower for $NO_3^-$ and more than 600 times lower for $PO_4^{3-}$ concentrations reported in the Katari River mouth (Duwig et al., 2014).
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This points towards a rapid assimilation of nutrients (especially phosphate) in just a few kilometers within the lake. The low concentration of nutrients along Cohana Bay, despite the anthropogenic contribution, is a clear indication of its rapid assimilation by microorganisms and algae, especially by the macrophyte-related periphyton, most of which is attached to *S. californicus* (totora) in the belts that this macrophyte forms inside the Cohana Bay.
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232 The great capacity of nutrient absorption by periphyton has already been documented in previous studies in this bay (Sarret et al., 2019; Quiroga-Flores et al., 2021). Hence, the natural mitigation of this anthropogenic contamination in the Cohana Bay results from (i) the uptake of DOC and nutrient by aquatic vegetation and algae and (ii) its dilution with pristine freshwater from the center lake region.
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237 In the coastal site of Bay of Quehuaya, an analog of Cohana Bay without wastewater discharge, water physicochemistry had intermediate values, similar to those found at CB 3. Such
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239 moderate eutrophication status is likely due to the low-intensity anthropogenic activities in the
240 surrounding villages (i.e., domestic wastewater, subsistence livestock farming and agriculture).

241 3.2. Carbon and nitrogen isotopic composition of Lake Titicaca's ecological compartments

242 3.2.1. Macrophytes: indicators of ecotopes and anthropogenic impact

243 Aquatic plant distribution in Lake Titicaca has previously been described to depend on water
244 column depth (Fig. 1c). About 60 % of the Lago Menor's surface bottom is covered by
245 aquatic vegetation (Collot et al., 1983).

246 Along the Cohana Bay transect, δ¹³Corg values of macrophytes (all species considered) rise with
247 the distance to the source along the eutrophication gradient (Fig. 3, top panel). The most
248 depleted values are in the very shallow areas near the Katari River outlet (CB 1; δ¹³Corg = -29.0
249 ± 0.4 ‰ and -26.5 ± 0.2 ‰ for the wet and dry season, respectively) in the range of reported
250 values for emerged macrophytes [δ¹³Corg = -26 ‰ to -28 ‰, green-colored band in Fig. 3,
251 Rowe et al. (2002), Miller et al. (2010)]. The highest ones are in deeper areas towards the open
252 lake (CB 6; δ¹³Corg = -7.5 ± 0.6 ‰ and -8.2 ‰ for the wet and dry season, respectively), typical
253 of the range reported for submerged macrophytes (ca. -12 ± 4 ‰, Fig. 3, dark cyan colored
254 band) of Lake Titicaca [Rowe et al. (2002), Miller et al. (2010), Guedron et al. (2020)]. Such
255 patterns of δ¹³Corg in macrophytes reflect the pathway of carbon uptake, which is related to the
256 plant's ecological distribution with water depth (e.g., emerged vs. submerged macrophytes), as
257 well as changes in the quality (trophic status) of the water column. In the very shallow coastal
258 sites, the dense vegetation of emerged macrophytes (i.e., S. californicus) which belongs to the
259 group of the helophytes, exhibit isotopic values (average: -26.4 ± 1.5 ‰) within the range of
260 terrestrial plants (-32 to -22 ‰), because most of the carbon is captured from the atmosphere
261 (i.e., CO₂; δ¹³C = -8 ‰) by the leaves. In contrast, submerged macrophytes (i.e., Myriophyllum
sp., *Elodea* sp., *Chara* sp., *Stuckenia* sp. and *Zannichellia* sp.) exhibit more positive δ¹³Corg values (average: -13.21 ± 6.5 ‰) because the carbon source mainly originates from the dissolved inorganic carbon (DIC), which includes dissolved CO₂ and all its pH-dependent forms. Hence, the difference in δ¹³Corg between these aquatic plants is mostly attributable to their inorganic carbon source. The boundary layer diffusion effects during atmospheric vs. aquatic photosynthesis decrease the discrimination against the heavy isotope, resulting in higher δ¹³Corg values in submerged macrophytes (Finlay and Kendall, 2007).

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<figure><img src="image_27.png" /><figcaption>**Figure 3.** δ¹³Corg (top) and δ¹⁵N (bottom) stable isotope composition of macrophytes (submerged: ∇, emerged: △) at Cohana Bay, the open lake and coastal sites (Q1 and Q2 = Bay of Quehuaya, HU=Huatajata). Colored triangles refer to wet (blue) and dry (red) season. Colored areas refer to: allochthonous terrestrial δ¹³C (brown), anthropogenic δ¹⁵N (yellow), submerged macrophyte δ¹³C from lake shallows (dark cyan), emerged macrophyte δ¹³C (green) and biological nitrogen fixation (BNF, cyan). Reference thresholds</figcaption></figure>

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of colored areas are as follows: allochthonous $\delta^{13}$C [-23 ‰, Finlay and Kendall (2007) and Chappuis et al. (2017)], anthropogenic $\delta^{15}$N [+10 ‰, McClelland et al. (1997) and Finlay and Kendall (2007)], typical submerged macrophyte $\delta^{13}$C [-8 to -16 ‰, Rowe et al. (2002)], typical emerged macrophyte $\delta^{13}$C [-26 to -28 ‰, Rowe et al. (2002)] and BNF [-1 to -3 ‰, Zhang et al. (2014)]. $\delta^{13}$C of the soil catchment (-23.8 ‰) is presented as a black dotted line. The three comparative sampling zones are indicated on the top of the figure with gray bands.

280

281 Amongst the species present in the Bay, emerged macrophytes (S. californicus) show similar
282 $\delta^{13}$C$_{org}$ values (average: -26.4 ± 1.5 ‰) regardless of their location and the season. In contrast,
283 submerged macrophytes collected in the coastal (Q2 and HU) and deep sites near the open
284 lake (CB 6 and L3) exhibited the highest $\delta^{13}$C$_{org}$ values, up to -7 ‰ (Fig. 3). Along the
285 Cohana bay, $\delta^{13}$C$_{org}$ of the submerged macrophytes decrease with distance to the KR inlet (-
286 27.4 ± 1.3 ‰ at CB 1 to -7.7 ± 0.5 ‰ at CB 6, seasonal average) likely resulting from changing
287 sources in the DIC pool. In submerged macrophytes near the Katari River inlet, $\delta^{13}$C$_{org}$ values
288 are similar to allochthonous inputs dominated by terrestrial organic matter (OM) from the
289 catchment (ca. -23.8 ‰, dotted line, Fig. 3). This indicates that the source of DIC for
290 submerged macrophytes close to the Katari mouth mainly originates from the recycling of
291 mixed terrestrial or anthropogenic OM. In contrast, toward the open lake, it rather originates
292 from locally produced carbonate dissolution and the decomposition of OM, mostly from
293 *Characeae* [-8 to -16 ‰, Rowe et al. (2002), Miller et al. (2010)].

294 Strikingly, nitrogen stable isotopes signatures of macrophytes exhibit a reverse trend compared
295 to C isotopes, with decreasing $\delta^{15}$N values with distance. The highest $\delta^{15}$N values are found in
296 the very shallow areas near the Katari River outlet, with higher values during the wet season,
297 i.e., between CB 1 ($\delta^{15}$N$_{wet}$ = +19.8 ± 1.8 ‰, $\delta^{15}$N$_{dry}$ = +8.9 ± 1.5 ‰) and CB 2 ($\delta^{15}$N$_{wet}$ =
298 +21.4 ± 2.0 ‰, $\delta^{15}$N$_{dry}$ = +10.7 ± 2.0 ‰) which are typical of N-enriched systems [i.e.,
299 between +10 and +20 ‰ (McClelland, Valiela et al. 1997), dark yellow band in Fig. 3. The
300 lowest δ¹⁵N values are found towards the open lake and remains in a narrow range
301 independent of the season (e.g., CB 10.7: δ¹⁵N = -3.6 ‰ ± 0.5 ‰, seasonal average), similar to
302 those reported by Miller et al. (2010), typical of N-depleted ecosystems [-1 to -3 ‰, cyan
303 colored band; Zhang et al. (2014]. The costal sites (Q1 = -0.51 ‰, Q2 = -0.2 ± 1.2 ‰, Hu = -
304 4.2 ± 5.6 ‰), and relatively deep (L3 = -0.7 ± 1.1 ‰) sites show similar low values.

305 In contrast to carbon cycling and isotopic signature (δ¹³Corg), the differences in the signature of
306 δ¹⁵N between macrophytes is caused by a wide and combined range of factors, including the
307 various metabolic pathways used for nitrogen assimilation, changes in the types of nitrogen
308 assimilated, and the relative activity of nitrogen-fixing and denitrifying bacteria (Handley and
309 Raven, 1992). Amongst these factors, the abundance of assimilated dissolved inorganic
310 nitrogen (DIN) by plants, which can be in the form of nitrate (NO₃⁻) and ammonium (NH₄⁺),
311 mainly depends on three major sources of supply; (i) the atmosphere (dry deposition and
312 biological fixation), (ii) the catchment (i.e., mineralization of the OM), and (iii) fertilizers,
313 animal or human waste (Finlay and Kendall, 2007).

314 In N-enriched ecosystems (e.g, human impacted environments), elevated δ¹⁵N values (+10 to
315 +20 ‰) are generally attributed to the removal of volatilized ammonia from human and
316 animal wastes during early stages of wastewater degradation (McClelland et al., 1997) as well as
317 the higher trophic level from which they have originated [i.e., consumers exhibit a 2 to 3 ‰
318 δ¹⁵N enrichment relative to their diet, Kendall et al. (2007)]. In contrast, in N-limited
319 ecosystems such as the open lake, nitrogen biological fixation (NBF) plays a major role in
320 supplying bioavailable nitrogen (i.e., NH₃) to the N pool with δ¹⁵N ratios [-1 to -3 ‰, Zhang et
321 al. (2014)] that are close to those of atmospheric N₂ (ca. 0 ‰). (Chappuis et al., 2017).
The low N concentration in the entire Lake supports the hypothesis of a rapid N assimilation in the water column despite the high anthropogenic discharge into the Cohana Bay, and confirms that N and P are limiting factors for aquatic biota within the lake [Fig. 2, and Dejoux and Iltis (1991)]. In N limited ecosystem, N uptake is expected to produce little net fractionation between the δ¹⁵N of DIN and macrophyte (Finlay and Kendall, 2007). Therefore, δ¹⁵N of macrophyte in Lake Titicaca likely integers the isotopic signature of the DIN source during the plant's life cycle. Although one cannot rule out significant fractionation of N associated with *in situ* growth and respiration (e.g., nitrification and denitrification) in the anoxic Katari river and the Cohana bay waters and sediment, high δ¹⁵N values in macrophytes at the mouth of the Katari River (up to +21‰, Fig. 3) supports manure piles and septic waste as the main anthropogenic N source. These values are consistent with δ¹⁵NNO₃⁻ values reported for the lower Katari basin surface and ground waters (Flores Avilés et al., 2022). Hence, δ¹⁵N values in macrophytes confirms physico-chemical observations (Fig. 2) and support anthropogenic inputs as the dominant N source within the first 5 km of the Cohana Bay, whereas depleted δ¹⁵N values outside this area support the fixation of atmospheric N as the major supplier to the DIN pool.

In addition, the large seasonal variations in δ¹⁵N observed within the first 5 km of the Cohana Bay reflect enhanced anthropogenic N inputs during the wet season, likely resulting from the leaching of urban and agricultural areas (Achá et al., 2018). In contrast, lower values of δ¹⁵N values in macrophytes at the mouth of the Katari River (+8.0 to 10.7 ‰, Fig. 3) during the dry season indicates an anthropogenic contribution mixed with a depleted ¹⁵N source.

### 3.2.2. Periphyton: an integrator of macrophytes and water column signal
Periphyton is defined as a biofilm composed of an assemblage of organisms (e.g., algae, bacteria, fungi, microinvertebrates, prokaryotes, and protozoa), and organic and inorganic detritus held in a mucopolysaccharide matrix attached to macrophytes (Lowe, 1996). Several studies performed in Lakes Titicaca and Uru-Uru reported the periphyton to act as a trap for suspended solids and dissolved elements, including nutrients (Alanoca et al., 2016; Lanza et al., 2017; Sarret et al., 2019; Guédron et al., 2020; Quiroga-Flores et al., 2021).

<figure><img src="image_28.png" /><figcaption>**Figure 4.** δ¹³C<sub>Chlak</sub> (top) and δ¹⁵N (bottom) stable isotope composition of periphyton at Cohana Bay, the open lake and coastal sites (Q1 and Q2 = Bay of Quehuaya, HU=Huatajata). Colored diamonds refer to wet (blue) and dry (red) season. Error bars represent confidence intervals for data at each site. Colored areas refer to: allochthonous terrestrial δ¹³C (brown), anthropogenic δ¹⁵N (yellow), submerged macrophyte δ¹³C from Lake Titicaca shallows (dark cyan), algae δ¹³C (pink) and biological nitrogen fixation (BNF, cyan). Reference thresholds of colored areas are as follows: allochthonous δ¹³C [-23 ‰, Finlay and Kendall (2007) and Chappuis et al. (2017)], anthropogenic δ¹⁵N [+10 ‰, McClelland et al. (1997) and Finlay and Kendall (2007)], typical submerged</figcaption></figure>
macrophyte δ¹³C [-8 to -16 ‰, Rowe et al. (2002)], local planktonic δ¹³C [-22.5 ‰, Rowe et al. (2002)] and BNF [-1 to -3 ‰, Zhang et al. (2013)]. δ¹³C of the soil catchment (-23.8 ‰) is presented as a black dotted line. The three comparative sampling zones are indicated on the top of the figure with gray bands. The star symbols (*) indicate the hosting macrophytes from which periphyton was taken (*: Chara sp.; **: Elodea sp. - Stuckenia sp. - Zannichellia sp.; ***: S. californicus - Stuckenia sp. - Zannichellia sp.).

In the entire set of periphyton samples (Fig. 4, top), δ¹³C<sub>bulk</sub> did not exhibit any specific spatial distribution trend during the dry season, whereas a gradual increase of δ¹³C values with distance was observed in the Cohana Bay during the wet season from CB 2 (δ¹³C<sub>bulk</sub> = -32.4 ‰) to CB 5 (δ¹³C<sub>bulk</sub> = -18.15 ‰). It is worth mentioning that samples collected during the wet season were exclusively associated with emerged macrophytes (i.e., *S. californicus*), whereas the ones collected during the dry season were associated with different macrophytes (Fig. 4). A previous study in shallow eutrophic lakes has reported that the δ¹³C of the periphyton closely reflects the isotopic composition of the macrophyte that serves as substrate (De Kluijver et al., 2015). Accordingly, the high seasonal variation at coastal site Q2 (11.3 ‰) confirms this effect, where the ¹³C-depleted value measured during the wet season is similar to that of the hosting macrophyte *S. californicus*, whereas the ¹³C-enriched value measured during the dry season matches that of the submerged macrophytes (ca. -12 ± 4 ‰, dark cyan colored band in Fig. 4).

In the case of Cohana Bay, the lowest δ¹³C<sub>bulk</sub> values (average: -26.2 ± 5.4 ‰) were measured during the wet season and had similar values to those of the ¹³C-depleted OM of emerged macrophytes, suggesting that the periphyton collected in the vicinity of the Katari outlet during the wet season have assimilated dominantly the OM of its hosting emerged macrophyte together with a contribution of entrapped or adsorbed allochthonous OM from the Katari River inlet. In contrast, periphyton collected towards the open lake (i.e., CB 5) and in Huatajata
381 (HU) have assimilated the ¹³C-enriched OM of the submerged macrophytes (ca. -12 ± 4 ‰,
382 dark cyan colored band in **Fig. 4**) and algae [ca. -22.5 ‰, pink-colored band in **Fig. 4**; Rowe et
383 al. (2002). The higher seasonal differences found for the shallow coastal sites at CB 2 (15.7 ‰)
384 compared to that of CB 3 (6.3 ‰) supports a significant contribution of allochthonous OM
385 during the wet season, which decreased with distance to the Katari outlet.

386 In contrast to δ¹³C signatures, the δ¹⁵N of periphyton exhibited a trend similar to their hosting
387 macrophytes, with decreasing values from +12 ‰ at CB 2 to +2.1 ‰ at CB 5 during the wet
388 season (10 ‰ difference) and from +10.3 ± 2.3 ‰ at CB 2 to +2.5 ± 2 ‰ at CB 3 during the
389 dry season (7.8 ‰ difference, bottom panel in **Fig. 4**). Such ca. 7.8 ‰ drop in δ¹⁵N in almost 3
390 km confirms the rapid assimilation of the anthropogenically-derived N followed by a
391 dominance of DIN supplied by BNF. Amongst coastal sites, Huatajata exhibited δ¹⁵N ratios
392 (+1.7 ± 0.8 ‰) typical of pristine or weakly polluted waters, whereas Bay of Quehuaya showed
393 slightly higher δ¹⁵N values (Q1 = 4.4 ‰, Q2 = 3.5 ‰) that could be attributed to a slight
394 contribution of animal waste N inputs during the wet season.

395
3.2.3. Suspended particulate matter (SPM) and sediment: spatio-temporal indicators

396 Lake suspended particulate matter (SPM) is composed of both autochthonous (i.e., debris of
397 primary producers and periphyton) and allochthonous organic matter (i.e., terrestrial OM from
398 the catchment) that settles to the bottom sediment. Therefore, both the SPM and sediment can
399 be considered as an integrative picture of the lake productivity at short and longer temporal
400 scale, respectively.

401 Similar to macrophytes, δ¹³C<sub>bulk</sub> values for SPM exhibited a linear increase with distance to the
402 KR in the Cohana Bay of 11.2 ‰ (upper panel in **Fig. 5**). However, these values remained
constant in the open lake and the coastal sites (average $\delta^{13}C_{bulk} = -17.7 \pm 0.8$ ‰ and -19.8 ± 0.9 ‰, for SPM and sediment respectively), and within the range of submerged macrophytes.
These observations support our earlier findings that the contribution of allochthonous and
anthropogenic OM inputs are significant within the eight first km within the bay. They also
imply a major anthropogenic OM contribution from upstream cities as the low $\delta^{13}C_{bulk}$ ratios
found for SPM at CB1 (-29‰) is typical of sewage effluents and catchment runoff [average
$\delta^{13}C_{org}$ = -24.2 ‰, Gearing et al. (1991), Rosenmeier et al. (2004)]. Further in the bay and at
other coastal sites, the decomposition of OM from submerged macrophytes (i.e., mostly Chara
sp.) is the largest supplier to the DIC pool with a small contribution from pelagic primary
productivity (i.e., algae).

Similar to SPM, $\delta^{13}C_{org}$ of sediment exhibit a gradual increase of 8.6 ‰ within the bay.
However, whereas the SPM exhibit very negative values close to the KR outlet, the ones of
sediment are in the typical range of soil and emerged macrophyte values and do not allow
attributing a significant anthropogenic contribution. Then, the gradual rise of $\delta^{13}C_{org}$ within the
bay can be attributed to the gradual change in dominant vegetation from emerged to
submerged macrophytes. It is worth mentioning that collected sediments covered the ten first
centimeters and have likely integrated the last 20 to 100 years of sedimentation. Indeed, the
average sedimentation rate ranges between 0.1 to 0.5 cm per year in the coastal sites of Lake
Titicaca (Guedron et al., 2020). Hence, the measured $\delta^{13}C_{org}$ signature integrates both the
historical sediment signature and possible historical change in ecological distribution related to
past lake-level changes (Guedron et al., 2021).

424
<figure><img src="image_29.png" /><figcaption>**Figure 5.** δ<sup>13</sup>C (top) and δ<sup>15</sup>N (bottom) stable isotope composition of SPM (●, dry season) and sediment (□) at each sampling site for Cohana Bay, the open lake (CH = Chua, Lm = Lago mayor) and coastal sites (Q1 and Q2 = Bay of Quehuaya, HU=Huatajata). Error bars represent confidence intervals for data at each site. Regressions are plotted only for p values < 0.05. Colored areas refer to: allochthonous terrestrial δ<sup>13</sup>C (brown), anthropogenic δ<sup>15</sup>N (yellow), submerged macrophyte δ<sup>13</sup>C from Lake Titicaca shallows (dark cyan), algae δ<sup>13</sup>C (pink) and biological nitrogen fixation (BNF, cyan). Reference thresholds of colored areas are as follow: allochthonous δ<sup>13</sup>C [-23 ‰, Finlay and Kendall (2007) and Chappuis et al. (2017)], anthropogenic δ<sup>15</sup>N [+10 ‰, McClelland et al. (1997) and Finlay and Kendall (2007)], typically submerged macrophyte δ<sup>13</sup>C [-8 to -16 ‰, Rowe et al. (2002)], local planktonic δ<sup>13</sup>C [-22.5 ‰, Rowe et al. (2002)] and BNF [-1 to -3 ‰, Zhang et al. (2013)]. δ<sup>13</sup>C of the soil catchment (-23.8 ‰) is presented as a black dotted line. The three comparative sampling zones are indicated on the top of the figure with gray bands.</figcaption></figure>

The δ¹⁵N values of SPM also mirrored those of macrophytes, showing a linear decrease (11.1 ‰) difference between CB 1 and CB 6) with increasing distance to the Katari river. As for δ¹³C,
the signatures of δ¹⁵N remained constant in the open lake and at the coastal sites with depleted values (average: δ¹⁵NSPM = +1 ± 1.2 ‰). Again, these δ¹⁵N values close to 0 in remote areas are indicative of nitrogen biological fixation [-1 to -3 ‰, Fig. 5, cyan colored band; Zhang et al. (2014)], most likely due to insufficient NO₃⁻ in the DIN pool (Gu et al., 2006), contrasting with values near the Katari River outlet (δ¹⁵N; CB 1 = +9.5 ‰, CB 2 = 12.2 ‰) where wastewater-derived N is the main source.

In contrast, δ¹⁵N values of the sediment did not show significant changes within the Cohana Bay, and remained in a narrow range of variation (2.6 ‰ difference) within the range of values of the BNF domain (δ¹⁵N = -1.3 ± 2.4 ‰) as for the open lake and the coastal areas. Such a different trend found in δ¹⁵N between SPM and sediment likely results, as for δ¹³Corg, from the large time period covered by the collected sediment cores. Hence, the recent anthropogenic signal has likely been smoothed or obliterated by the pristine historical signal of the sediment.

### 3.2.4. Fingerprinting anthropogenic contamination with δ¹³C vs δ¹⁵N

The spatial changes in both δ¹³C and δ¹⁵N signatures for macrophytes, SPM, and to a lesser extent for periphyton within the Cohana Bay, reveal that anthropogenic contamination is progressively attenuated within the bay. In contrast, other coastal sites (i.e., Quehuaya Bay and Huatajata) distant from the Katari Outlet only show a low contamination signal.

At the seasonal scale, both the macrophyte and periphyton show increased spreading of the anthropogenic contamination within the bay during the wet season, a period of enhanced catchment leaching (i.e., soils and urban area), river discharge, and transfer of dissolved and particulate C and N to the Lake. In addition, the significant inter-seasonal lake-level changes, up to 2 meters (SENAMHI, 2021), also affect the ecotope distribution and the development of
461 specific plant species aggregates more or less tolerant to high nutrient loads depending on the
462 season and eutrophication status.

463 When using 2D isotopic graphs (Fig. 6), the differences are clearly noticeable for the spatial
464 changes between δ¹³C and δ¹⁵N signatures within each compartment. Indeed, the absence of a
465 significant correlation between δ¹³C and δ¹⁵N suggests that carbon and nitrogen recycling
466 processes are significantly different inside the bay. Regardless of the season, the δ¹³C of
467 macrophytes (except S. californicus), periphyton and SPM within the first 8 Km (i.e., between
468 CB 3 and CB 4) exhibit lower values (Fig. 6) typical of allochthonous C [Finlay and Kendall
469 (2007), Chappuis et al. (2017)].

470 In contrast, the δ¹⁵N of the four lake compartments depict a clearer pattern of δ¹⁵N depletion
471 from anthropogenic δ¹⁵N-enriched values, to lower ones typical of N-biological fixation
472 and/or atmospheric deposition around 5 km (Fig. 6). This rapid, close to the source, depletion
473 of heavier N isotopes likely indicates a rapid consumption of anthropogenic N in this N-
474 limited aquatic system, whereas the larger extension of the anthropogenic ¹³C signal supports
475 slower incorporation of anthropogenic C in the oligotrophic part of the system.
<figure><img src="image_30.png" /><figcaption>**Figure 6.** δ¹³C vs δ¹⁵N biplots of: a) macrophytes (submerged: ∇, emerged: △), b) periphyton (◇), c) sediments and particulate organic matter (□ and ○, respectively) for Cohana Bay and the coastal sites (Huatajata and Bay of Quehuaya). Symbols represent individual values of each sample evaluated in this study. Colors refer to: the coastal sites (blue) and the polluted (red), transitioning (yellow) and pristine or weakly polluted areas (green) within Cohana Bay. Huatajata and Bay of Quehuaya are presented as x-hair and crossed symbols, respectively. Colored dotted lines refer to: allochthonous terrestrial δ¹³C (brown) and anthropogenic δ¹⁵N (dark yellow). Reference thresholds of colored areas are as follows: allochthonous δ¹³C [-23 ‰, Finlay and Kendall (2007) and Chappuis, Serin, et al. (2017)] and anthropogenic δ¹⁵N [+10 ‰, McClelland, Valiela et al. (1997) and Finlay and Kendall (2007)].</figcaption></figure>

Hence, three zones can be identified in the Cohana Bay: (i) the first 5 km to the Katari River outlet where anthropogenic C and N inputs dominate, (ii) a transition zone between 7 and 8

477
478

479

480

481

482

483

484

485

486

487
488 Km from the KR outlet that marks the mixing and dilution of urban and terrestrial OM, and
489 (iii) a pristine zone in the deepest part of the bay dominated by N-fixation and autochthonous
490 C similar to the rest of the open lake, providing the biogeochemical isotopic baseline signature
491 of the ecosystem. The larger attenuation occurs within the belt of totoras which acts as a
492 physical barrier where the biological pump (dominated by periphyton) consumes the
493 discharged nutrients and entraps floating SPM.

494 Moreover, the 3 studied compartments showed substantial differences along the
495 eutrophication gradient. Submerged macrophytes and SPM accurately illustrated the dilution
496 between anthropogenic and natural OM sources. SPM illustrates greatly the two distinct
497 anthropogenic and pristine pools, with few intermediates. For macrophytes, the ecological
498 distribution along the bay shows the distinct ability of aquatic plant species to integrate the C
499 signal depending on the plant type, water depth and quality. Indeed, whereas the emerged
500 macrophyte (i.e., S. californicus, only found in very shallow areas) does not allow the C source
501 identification because of its terrestrial-like behavior (i.e., atmospheric C incorporation), the
502 submerged macrophytes, like Chara sp. and Elodea sp., which are relatively well distributed
503 along the transect, provide a large range of δ¹³Corg. This is not the case for myriophyllum sp.,
504 which is only encountered in the pristine coastal site and may indicate its sensitivity to
505 eutrophication. In contrast, both macrophytes (emerged and submerged) exhibit high
506 differences in δ¹⁵N signatures as they integer DIN via the aqueous media.

507 Periphyton C and N isotope signatures range in between the ones of macrophytes and SPM.
508 Their δ¹³C values are however tightly related with those of their hosting plant (Figs. 6a and
509 6b), whereas their δ¹⁵N is as narrow as the one of SPM (Figs. 6b and 6c) with all samples
having an atmospheric N₂ source (i.e., close to 0) except for those located within the first 5 km from the KR.

Finally, for other shallow coastal sites away from Cohana Bay (i.e., Huatajata and Bay of Quehuaya), the absence of a significant pollution signal indicated by their δ¹³C and δ¹⁵N isotopic signatures reflects their overall intact status. However, some moderately higher values indicate that agricultural land leaching is the main source of allochthonous OM during the wet season.

### 3.3. Significance of lake ecological compartments as bioindicators of eutrophication

#### 3.3.1. Mixing model with δ¹³C vs δ¹⁵N

To estimate the contribution of wastewater effluent as a source of C and N to the ecosystem, we used a linear mixing model to calculate plant uptake of wastewater-derived carbon and nitrogen (Cecchetti et al., 2020). In the absence of precise isotopic data for each anthropogenic source (e.g., human and animal sewage, synthetic fertilizers...), a simplified two-source linear mixing model (taking into account a natural and an anthropogenic pool) was applied to our data according to Eqs. (1) and (2) for carbon, and (3) and (4) for nitrogen:

$$ \delta^{13}C_{\text{Plants}} = (\delta^{13}C_{\text{prist}}) * (f_{\text{prist}}) + (\delta^{13}C_{\text{WW}}) * (f_{\text{WW}}) \quad (1), \text{ and } 1 = f_{\text{prist}} + f_{\text{WW}} \quad (2) $$

$$ \delta^{15}N_{\text{Plants}} = (\delta^{15}N_{\text{atmos}}) * (f_{\text{atmos}}) + (\delta^{15}N_{\text{WW}}) * (f_{\text{WW}}) \quad (3); \text{ and } 1 = f_{\text{atmos}} + f_{\text{WW}} \quad (4) $$

where δ¹³CPlants, δ¹³Cprist, δ¹³CWW are the carbon isotope signatures of plants, pristine DIC and wastewater, and δ¹⁵NPlants, δ¹⁵Natmos, and δ¹⁵NWW are the nitrogen isotope signatures of plants, atmosphere and wastewater.
As mentioned previously, emerged and submerged macrophyte have different baseline $\delta^{13}$C$_{org}$ values because the fractionation of $^{13}$C$_{org}$ in macrophytes is plant-specific (i.e., pathway of DIC or atmospheric CO$_2$ uptake). Hence, to estimate the fraction of carbon uptaken without a single contribution of atmospheric CO$_2$, we only considered *Characeae* as their source of DIC may largely depend on the loading of DIC from the watershed (Bade et al., 2004), and because these submerged macrophytes have the largest distribution within the study sites. Carbon isotope endmember value for anthropogenic source ($\delta^{13}$C$_{WW}$) was taken from the one of SPM collected at the inlet of the Katari in Lake Titicaca (CB1; $\delta^{13}$C = -28.8 ‰). The pristine $\delta^{13}$C-DIC reference value ($\delta^{13}$C$_{prist}$) was taken from average values published for $\delta^{13}$C-DIC (-8.0 ‰) at the isotopic equilibrium with the atmosphere in alkaline and slightly positive pH (~8) lakes (Bade et al., 2004), which is similar to the values found in Characeae at site CB 6 and L3, and those reported for characeae in Lake Wiñaymarca (Miller et al., 2010).

Nitrogen isotope endmember value for wastewater $\delta^{15}$N was taken from Hydrocharitaceae samples collected during the most impacted wet season at the inlet of the KR (CB 1; $\delta^{15}$N = 23.3 ‰) which is in the range of reported $\delta^{15}$N for wastewater (10 to 20 ‰), and to the one of $\delta^{15}$N in nitrate of the Katari wastewater ($\delta^{15}$N = 19.2 ‰) from the upper part of the catchment (Flores Avilés et al., 2022). The atmospheric endmember ($\delta^{15}$N$_{atmos}$) was taken from the average nitrogen biological fixation (NBF = -3 ‰) reference value (Zhang et al., 2014).

The fraction of characeae biomass C contribution from wastewater to plants are represented in figure 7. Although the $^{13}$C mixing model could not be applied within the first five km of the bay because of the absence of Characeae in this area, the model suggests that the wastewater contribution is still visible between seven and eight km, and in the coastal site of HU which also receives wastewater. In contrast, the anthropogenic contribution appears negligible after
eight km from the KR outlet as well as in the rural coastal sites of Q1 and Q2 (Fig. 7A). It must be considered that this model is imperfect as it doesn't take into account biological productivity and respiration, which could induce important changes in DIC carbon isotope ratios depending on the different levels of metabolism along the slopes of the lake.

558

<figure><img src="image_31.png" /><figcaption>Figure 7. δ¹³Corg (top) and δ¹⁵N (bottom) stable isotope mixing model of macrophytes at Cohana Bay, the open lake and coastal sites (Q1 and Q2 = Bay of Quehuaya, HU=Huatajata). Colored diamonds refer to wet (blue) and dry (red) season. Error bars represent confidence intervals for data at each site. The three comparative sampling zones are indicated on the top of the figure with gray bands.</figcaption></figure>

559

560

561

562

563
The fractional contribution of biomass N from wastewater nitrogen to plants highlights a gradual decrease after the first five km from the shore, where the wastewater always exceeds 40% to more distant areas where they drop to around 10%, even at HU. The most striking feature illustrated by the model is the sharp uptake of wastewater N during the wet season which reaches up to 80 % within the first 5 km of the bay, whereas it stands between 40 and 50% during the dry season (Fig. 7B).

## 4. Conclusions and perspectives

The study of δ¹³C and δ¹⁵N signatures within various baseline compartments of Lake Titicaca show the different trophic status of the lake with areas greatly impacted by anthropogenic discharges that have low δ¹³C and high δ¹⁵N signatures whereas pristine areas have high δ¹³C and low δ¹⁵N signatures. The gradual change in both δ¹³C and δ¹⁵N signatures along the Cohana Bay transect are thus essentially driven by the change of anthropogenic and background/biogeochemical natural sources. This change greatly illustrates the assimilation of anthropogenically derived OM in this ecosystem, mostly attributed to the strong fixation of nutrients by endemic macrophytes and their periphyton. The combined use of δ¹³C and δ¹⁵N brings complementary information, with δ¹³C reporting about the C sources and recycling in the ecosystem, whereas δ¹⁵N provides information about the N sources and ecological productivity.

SPM and macrophytes (except for the δ¹³C of totoras) are the most suitable compartments for tracking the dissemination of anthropogenic discharges in the lake because their δ¹³C and δ¹⁵N compositions accurately reflects the trophic status of the water column. The δ¹⁵N signature of the periphyton is also a suitable indicator of wastewater inputs, but their δ¹³C signature shows
587 limitations due to its closeness to the δ¹³C values of the hosting macrophyte. As a large-time
588 integrator, the sediment may also be a suitable indicator of trophic changes in the lake.

589 Hence both δ¹³C and δ¹⁵N signature in macrophytes and SPM, together with punctual physico-
590 chemical measurments indicate that anthropogenically induced eutrophication is marked within
591 the five first kilometers to the Katari river mouth, followed by a rapid attenuation or auto-
592 epuration within the next 2 to 4 km resulting from the assimilation of discharged nutrients.

593 The results presented here bring new insights that are of great interest for pollution diagnostic
594 studies and remediation strategies. For instance, filtering artificial wetlands that take advantage
595 of the periphyton capabilities as a trap of organic matter, nutrients and heavy metals can be a
596 much-needed alternative for preserving such a fragile ecosystem in front of the growing
597 human pressure.

598

599 Aknowledgments

600

601 This work is a contribution to the EUTITICACA project (founded by the Impuestos Directos
602 a los Hidrocarburos IDH administrated by the Universidad Mayor de San Andrés, PI: D.
603 Acha: darioacha@yahoo.ca), COMIBOL project (INSU CNRS/IRD EC2CO Program, PI: D.
604 Point: david.point@ird.fr) and LA PACHAMAMA project (ANR CESA program, No ANR-
605 13-CESA-0015-01, PI: D. Amouroux: david.amouroux@univ-pau.fr).

606 We wish to thank, J. Gardon, A. Terrazas, C. Gonzalez, N. Clavijo, L. Salvatierra, R. Rios, J.C.
607 Salinas, A. Castillo, M. Claure, J. Tapia, J.L. Duprey (IRD Bolivia), la Familia Catari (Don
608 Ramon, Don Maximo, Don Eric, Don Ruben and Donia Maria) and Don German Calizaya
609 (Fishermen Association, Machacamarca, Bolivia) for their help and assistance during the field

610 campaigns.
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