Marine Algal Response to Cultural Eutrophication in a Tidal System in Argentina

Abstract

Cultural eutrophication caused by human activity significantly impacts benthic ecosystems. This study investigated how different phytobenthic components—rhodophyte germlings, mesoalgal and macroalgal assemblages, and Ulva cf. lactuca—respond to nutrient enrichment in a tidal channel system in San Antonio Bay, Argentina. Two experiments were conducted: one in spring examined the interaction between nutrient enrichment (N + P, N + P + Fe) and grazing pressure on early and established algal communities, and the other in autumn assessed nutrient effects on assemblages and Ulva cf. lactuca. Results showed that early successional stages, such as germlings and mesoalgae, responded most strongly to nutrient inputs, while mature macroalgae remained largely unaffected. Significant growth of mesoalgae, with increased pigment concentrations (chlorophyll a, c, and carotenoids), occurred at the eutrophied SAO Channel in spring. Nutrient additions increased rhodophyte germlings but eventually reduced diatom-dominated mesoalgal growth. Mature macroalgae showed site-specific differences but did not respond to fertilization. Grazing effects were evident in treatments with protective cages, suggesting herbivory influences early-stage algal development. Overall, the study emphasizes the importance of the successional stage, grazing pressure, and environmental nutrient history in shaping benthic algal responses to eutrophication, offering key insights into the dynamics of coastal ecosystems under increasing nutrient stress.

1. Introduction

The anthropogenic release of nutrients is of increasing concern for the functioning of coastal ecosystems worldwide [1,2,3]. Starting with a pulse of nutrients, which can initiate blooms of different primary producers, such as phytoplankton and benthic algae [4], and is often followed by organic matter degradation, oxygen depletion and concomitantly the death of several organisms (e.g., fishes and crustaceans), the process of cultural eutrophication can modify the entire coastal ecosystem [5,6,7]. Such degradation of coastal ecosystems can impact economic activities such as tourism and fisheries and may have severe consequences on human health [8]. Moreover, human-driven eutrophication often leads to a misbalance in natural nutrient levels, mainly due to the increased anthropogenic influx of N and P (e.g., fertilizer release, sewage disposal) [9].

With the aim of preventing the escalation of these negative effects, much scientific research and coastal ecosystem management efforts have been directed towards understanding eutrophication processes, with a focus on early detection [10]. In this sense, the rapid response of micro- and macroalgal blooms to changes in nutrient levels has been shown to be a valuable indicator of eutrophication [11,12,13].

Benthic micro- and macroalgae are ubiquitous ecological key groups within coastal ecosystems. For instance, they serve as a food source for several organisms [14,15], and form microhabitats for demersal fishes, invertebrates and microorganisms [11,16,17]. Due to their mat-like growth, e.g., of bloom-forming Ulva species or tube-dwelling diatoms, they can alter velocity profiles and stabilize sediments [5,18,19]. Due to their high species number, e.g., over 12,000 species of macroalgae (e.g., Phaeophyceae > 2000, Florideophyceae > 7.000, Chlorophyceae > 3900) [20], they play an essential role in terms of biodiversity [14] and participate in the nutrient cycling [21]. There is also commercial interest in algal biomass from a variety of different industries, and algal cultivation is even considered to provide a valuable tool to mitigate climate change alterations [22]. Thus, a better understanding of algal responses to elevated nutrient levels will not only help to detect eutrophication but will also help to understand and predict ecological consequences in a much broader sense. Next to the essential macro nutrients nitrogen and phosphorous, other trace elements, like iron, play essential roles in the physiological metabolism and enzymatic reactions of photosynthetic organisms (e.g., for respiration or nitrogen assimilation) [23]. As iron is considered a limiting element in algal growth [24], an artificial addition of iron to regulate algal blooms and precipitate phosphorous is a common restoration approach applied for different aquatic habitats [25,26].

There is evidence that benthic successional trajectories change under different nutrient and herbivory conditions. A number of studies simulating the onset of overfishing (e.g., by reducing grazing pressure) in combination with eutrophication (e.g., increased nutrient inputs) have repeatedly shown a shift in dominance from slow-growing calcareous to more opportunistic filamentous algal taxa in hard-bottom ecosystems [27,28]. Under this misbalanced ecological scenario, especially opportunistic bloom forming, but also invasive species can play a crucial role, profiting from the available resources, quickly expanding in biomass and range [29,30]. Next to taxa identity, seasonality can also play an important role, as shown in combined grazer enclosure–nutrient enrichment experiments by [31]. Here, significantly higher standing stocks in coral reef algae were reached under grazer exclusion in nutrient-enriched conditions only during the warmer spring season, whereas no differences were observed during the colder season. Given the known seasonal changes in algal size and composition, it can be expected that different grazers favor the benthic algal communities and, consequently, the associated grazer identities might change over time.

The Argentine Patagonian coast is composed of a mosaic of several almost pristine sites mixed with a few places subject to locally increased anthropogenic pollution [32,33,34]. The San Antonio Bay (S40°43, W64°56, Argentina) is a good example of coastal changes related to increases in human population. The area shows high eutrophication rates, with nutrient concentrations similar to those found in the polluted central basin of the Italian Venice lagoon [12]. As a consequence of the macro tidal regime of about 9 m, the bay experiences a large daily water exchange that partially relieves the land-derived N loads, as well as the accumulation of biological products [32,35,36]. Despite the daily water flushing, nutrient concentrations remain in the system long enough at low tide to support high biomass and diversity of macroalgae near the town of San Antonio Oeste [17,32].

Close to the town of San Antonio Oeste, two tidal channels with contrasting nutrient and grazer loads have been intensively studied in terms of their algal and invertebrate dynamics (e.g., Martinetto et al., 2010, Fricke et al., 2016) [11,32]. The continuous nutrition and oxygenation favored the presence of persisting macroalgal stocks, inhabited by various invertebrate consumers [36]. These macroalgae seem to play a crucial role in the associated marine ecosystem, as spores of Ulva cf. lactuca have been shown to dominate the mesoplankton in the bay during spring [35]. Consequently, the relative strength of these observed bottom-up (high nutrient input) and top-down (high grazer presence) forces plays a crucial role in shaping the coastal environment. Despite these findings, the effects of nutrient pulses on successional trajectories across different life stages remain unclear. Furthermore, the balance between top-down and bottom-up regulatory forces at various stages presents another key question that merits further exploration.

To better understand the underlying benthic dynamics, a former study [11] followed the natural benthic succession in the different channels over one year. This study showed strong differences in phytobenthic composition between the channels throughout their succession. Differences were observable already after 3 days in the initial biofilm community, followed by differences in the rather dense growing mesoalgal and later more complex macroalgal communities.

With the aim of identifying potential indicator taxa and studying possible nutrient thresholds for the envisaged ecosystem, the present study conducted a multifactorial in situ experiment in the well-studied bivalent channel system. Simulating the increased anthropogenic nutrient influx by a controlled release of two different mineral compositions (N + P and N + P + Fe) in the pristine and the eutrophied channel, the study tested the potential response of different phytobenthic successional stages (e.g., germlings, mesoalgal, macroalgal communities and grown foliose Ulva thalli). In addition, the potential effect of grazer abundance was investigated at the beginning of the growth season in spring with a cage enclosure experiment.

We hypothesized that a pulse of nutrients at different successional stages will lead to different phytobenthic assemblages, and the outcome will also be affected by the combination of nutrient composition, grazer abundance and site identity.

2. Materials and Methods

2.1. Research Area and Experimental Sites

Field work was conducted in the San Antonio Bay in Northern Patagonia (Argentina, S40°43, W64°56). The region is characterized by strong seasonality and daily environmental alterations, due to the influx of Patagonian wind systems and strong tidal activities [37,38]. In addition, driven by climate change, further alterations are becoming visible, with a predicted increase in extreme rain events for the area [39]. To study the potential effect of increased human nutritional influx on the coastal ecosystem, a macro tidal system amplitude up to 9 m, and strong currents (2 m/s) have been chosen. Within this system, two tidal channels with contrasting nutrient and grazer loads were used to set the experiments. Sourced by the same tidal inlet, these channels have been used in previous studies to evaluate the effects of eutrophication [11,15,40]. The tidal channel running alongside the town of San Antonio Oeste (hereafter referred to as the SAO Channel) is subject to the constant impact of cultural eutrophication, resulting in frequent algal blooms [12,15]. In contrast, a parallel channel that runs farther from the human population (hereafter CONTROL channel) shows much lower nutrient loads [12,32,40]. During the study, the CONTROL channel was mainly covered by macroalgae growing attached to pebbles buried in the sediment. The red algal order Ceramiales dominated the site, with Polysiphonia as the most abundant genus [11]. In contrast, the SAO channel showed a high abundance of members of the green algal family Ulvaceae, forming a standing bloom, as described by Martinetto et al. (2010) [32]. Two experimental sites, CONTROL (S40°43.19 W64°56.52) and SAO (S40°43.62 W64°56.80), were defined at each channel (Figure 1A).

2.2. Experimental Set-Up

2.2.1. Phytobenthic Units

Considering the different succession and development stages observed, we distinguished four different types of phytobenthic algal units, grown on comparable hard substrates, proven as suitable settlement substrate in former studies [18,41,42,43].

GERMLING: In the initial stage, the settlement and recruitment of spores and germlings play a crucial role in the life cycle of benthic macroalgae. To investigate the identity of early macroalgal settlers, pieces (1.5 × 2 cm) of polyethylene terephthalate (PET, Melinex^®, Plano, Wetzlar, Germany) were applied as settlement substrates [18,41].

MESO: Forming dense and patchy assemblages, composed of a variety of small macroalgae, filamentous cyanobacteria and colony-forming diatoms, mesoalgae are a key group in shallow coastal ecosystems [44]. To allow the growth of early (12 to 20 days old) mesoalgal communities, unglazed ceramic tiles (2.5 × 6 cm) were used as substrata, as performed in other studies before [42,43].

MACRO: As primary producers and habitat shapers, grown macroalgae play different important ecological roles for the marine system. Macroalgal-dominated communities grown on unglazed ceramic 3 × 7.5 cm tiles previously exposed in a colonization set-up, horizontally to the water surface ~30 cm below low-tide water level and 20 cm above sediment, approximately four weeks prior to the two experiments (for Exp 1: 10 September 2012 and for Exp 2: 10 February 2013) at the corresponding experimental site (SAO, CONTROL) [11].

ULVA: In addition, one of the most abundant green algal genera, Ulva, which is intensely studied as an indicator for eutrophication in different studies [12,45], was chosen to be integrated in the experiment run in autumn as a fourth phytobenthic unit. For the experiment, a foliose type Ulva cf. lactuca, grown on small stones, was collected in the SAO channel. Without separating the algae from the stones, thalli were spread on a white board, photographed and an individual area was calculated using image processing software ImageJ2 version 2.0.0 [46]. Thirty ULVA units were randomly distributed between the different treatments on 15 March 2013. The supporting stones were glued with epoxy on marked ceramic 3 × 7.5 cm tiles.

2.2.2. Nutrient Enrichment Diffusers

To manipulate the ambient conditions and study the potential impact of nutrient enrichment, we used granulated slow-release fertilizer (Profertil), a fertilizer commonly used in benthic ecology studies [31,47]. Nutrient enrichment diffusers (NDs) were constructed of white plastic flower pots (d = 20 cm). Each ND was closed by a plastic lid (d = 22 cm), perforated by a fixed pattern of 42 holes (d = 0.5 cm) that allowed the fixation of different experimental units (Figure 1B). The fertilizer was set in the middle of the pot in an enrichment capsule. Each capsule consisted of a perforated plastic flask (d = 5 cm, h = 5 cm) to allow the diffusion of nutrients and was closed by a screw cap for easy refill. Each diffuser was loaded with stones and sediments taken from the area, buried, leaving 5 cm above the sediment and fixed by rope and hooks to the sediment. All units were placed in situ, keeping similar water levels. Each ND was equipped with the whole combination of the four different phytobenthic units: GERMLING, MESO, MACRO, and ULVA (Figure 1C). The settlement substrata GERMLING, MESO and MACRO were placed horizontally to the water surface in the NDs. The experiment had three nutrient treatments: (i) ambient nutrient conditions (AMB, no nutrient addition), (ii) N and P enriched treatment (N + P), adding 20 g of Diammonium phosphate (18 N-46 P) and (iii) N-, P- and Fe-enriched treatment (N + P + FE), adding 10 g DAP + 10 g Iron (II) sulfate (Profertil, Bahia Blanca, Argentina). In order to keep a consistent nutrient elevation through the experiment, 20 g of fertilizer was added to each enrichment capsule every three days.

2.2.3. Macrofauna Exclusion Treatment

To test the potential impact of macrofauna, like the highly abundant borrowing crab Neohelices granulate [48], on the different phytobenthic units, a cage treatment was applied in the first experiment (EXP1). For this, cages (60 × 30 × 30 cm) constructed of green plastic wire (mesh size = 1 cm²) and fixed to the bottom by concrete reinforcement bars (20 cm) were placed over the NDs. A total of three exclusion treatments were performed: (i) none-caged open treatment (ambient), (ii) completely caged treatment (caged) and (iii) partly caged treatment (ca. 35%; cage control) as cage control to test for cage effect.

2.2.4. Experimental Plan and Sampling Processing

As in temperate regions, seasonal variation is a key driver of ecological succession [49], our experimental design incorporated seasonality as a central factor. Thus, two experimental runs were performed at the beginning (Experiment 1 (EXP1) in spring; 12 October to 1 November 2012) and at the end (Experiment 2 (EXP2) in late summer: 15 February to 27 March 2013) of the phytobenthic growth season (e.g., defined by the overall macroalgal cover in Martinetto et al., 2010 [32]). A summary of the experiments is presented in

Table 1. Overview of the different experiments (EXP1, EXP2), showing the exposure time at the two different sites (SAO and CONTROL) of the four phytobenthic units (GERMLING, MESO, MACRO, ULVA), exposed to different the factors Nutrients (NUT; AMB, N + P, N + P + Fe) and grazer exclosure (CAGE; open, caged, cage control). Replication numbers and total number of applied nutrient diffusers (ND) are provided for each experiment.

Site	Time (Days)	Unit	Factors	Levels	Replication	NDs
EXP1: Spring season
SAO	5	GERMLING	NUT X CAGE	7: AMB (open, caged, cage control); N/P (open, caged); N/P/Fe (open, caged)	6	42
SAO	20	MESO, MACRO	NUT	3: AMB, N/P, N/P/Fe	8	24
EXP2: Late summer season
SAO and CONTROL	12	MESO	NUT	3: AMB, N/P, N/P/Fe	5	15
	14	MACRO, ULVA	NUT	3: AMB, N/P, N/P/Fe	5	15

and Figure 1D.

EXP 1: Testing the Effect of Eutrophication and Grazer Exclusion in the SAO Channel

Bottom-up and top-down effects on GERMLING, MESO and MACRO were investigated in experiment 1 (EXP1) during spring (October 2012) for a period of 20 days within the eutrophied SAO channel. For this purpose, a full factorial experiment was carried out with the following seven treatments: (i) AMB + open, (ii) AMB + caged, (iii) N/P + open, (iv) N/P + caged, (v) N/P/FE + open, (vi) N/P/FE + caged and (vii) AMB + cage control. Each treatment was replicated 6 times, leading to a total of 42 NDs randomly deployed in the field in a zigzag pattern (2 m individual distance) and fixed to the bottom ~40 cm below the low-tide water level (Figure 1E). After six days, all cages were removed from the NDs due to the strong tidal currents that made it impossible to keep them fixed to the sediment. Consequently, the combined caged and eutrophic conditions in the SAO channel were studied only on the sequential succession of GERMLINGS, with samples taken after three days (T1 = 16 October 2012) and six days (T2 = 19 October 2012), while MESO and MACRO units, which were sampled at the end of the experiment, were only investigated for the effects of eutrophication. Thus, we processed for MESO and MACRO four of the six replicates originally caged and four of the six not-caged replicates. After testing for potential effects after the first six days under caged/no caged treatment and not finding differences (see

Table A3. Differences in rhodophyte GERMLINGS in response to the factors grazer exclosure (cage; open, half caged, caged) and succession time (T1, T2) based on analyses of variance (ANOVA). Significant differences are printed in bold. Conclusion refers to the outcome of Levene’s test, indicating each significant difference, indicated in bold.

		Recruit Numbers			Biovolume
Factor	df	MS	F	P	MS	F	p
(a) Ambient Conditions: Succession and Cage
Cage (C)	1	0.528	0.158	0.854	3 × 1011	0.608	0.551
Time (T)	2	13.444	4.033	0.054	5 × 1012	9.433	0.005
C × T	2	4.361	1.308	0.285	6 × 1011	1.172	0.324
Conclusion					S2 > S1
(b) S1: Cage and Nutrients
Nutrient (N)	2	5583	1442	0.252	1 × 1010	0.859	0.434
Cage (C)	2	0.028	0.007	0.933	3 × 1010	2158	0.152
N × C	2	2694	0.696	0.507	5 × 1010	3047	0.062
Conclusion
(c) S2: Cage and Nutrients
Nutrient (N)	2	35,583	5369	0.010	4 × 1011	0.582	0.565
Cage (C)	2	26,694	4028	0.054	4 × 1012	5886	0.021
N × C	2	9028	1362	0.272	3 × 1011	0.442	0.647
Conclusion		N/P/Fe > AMB			Open > Caged

), we pooled all eight replicates. This arrangement had the purpose of increasing the number of replications without compromising the time of processing.

EXP2: Testing the Effect of Eutrophication in the SAO and CONTROL Channels

In Experiment 2 (EXP2), during autumn (March 2013), MESO, MACRO and ULVA were exposed in both the eutrophic SAO and the pristine CONTROL channels (Figure 1F). NDs were fixed to the bottom ~30 cm below low-tide water levels using the same zigzag random pattern in SAO as in the spring experiment (EXP1) to allow seasonal comparisons. Also, in the CONTROL channel, this exposure pattern was applied, and NDs were set to comparable water depths.

MESO samples were sampled after 12 days (27 March 2013) while MACRO and ULVA were sampled 2 days later at the end of the experiment (29 March 2013). For the ULVA exposed in the CONTROL channel, the experimental run had to stop after a week (22 March 2013), due to the loss of individuals by tidal action. To investigate potential alterations in the composition of MACRO, additional MACRO tiles (n = 3) were taken from the colonization set-ups of the two channels at experimental start and end, and investigated for their taxa composition.

2.3. Sample Processing

During sampling events, each GERMLING sample was carefully transferred to a 10 mL flask filled with ambient seawater, whereas each MESO, MACRO and ULVA (with its supporting ceramic tile) was transferred into individual plastic bags filled with ambient water. All samples were transported to the laboratory (<1 h) in an opaque container. GERMLING samples were fixed as semi-permanent slides using 50% Karo corn syrup, preserved with 4% formaldehyde. The biomass of each MESO unit was removed using a razor blade and split into two parts. One part (1.5 × 3 cm) was immediately stored under −20 °C until analyses. A second part was dried to constant weight at 60 °C to quantify dry weight and calculate the community growth under the different experimental conditions. Each MACRO was divided into three parts: (i) 1.5 × 3 cm was removed from the tile and immediately frozen (−20 °C), (ii) 1.5 × 3 cm was removed and dried at 60 °C to determine dry weight and (iii) the remaining biomass was preserved with the tile in 4% formaldehyde for later microscopic analyses. For ULVA, the thallus area was determined following the same digital protocol as for the initial conditions. Tissue material was removed from thalli using a cork borer (d = 1 cm). Depending on the thalli size, n = 4 to 6 samples were taken and stored at −20 °C until further analysis.

2.4. Environmental Data

For EXP 1 (spring season), environmental data (water temperature, pH and salinity) were measured on 27 October and 1 November 2012 using the multi-sensor device HORIBA U10 (Horiba, Kyoto, Japan) in two different channels (sites). During EXP 2 (late summer season), temperature was hourly logged from 15 February to 27 March 2013, using temperature loggers (Intec Electrónica, Buenos Aires) exposed in the subtidal at the two experimental sites. To investigate potential differences in water motion between the different sites, and to investigate for potential treatment artefacts, clod cards (2.5 × 4 × 1.5 cm) made of plaster of Paris (Doty 1971) were exposed for three days in autumn (25–28 March 2013) during EXP2 at the colonization set-up and in additional NDs at both channels (n = 3). Relative differences in flow rates (C) between the different channels were calculated by C = te/me, where te refers to the measured weight loss of the clod cards deployed in the channels and me refers to the measured weight loss of the aquarium control treatment. To evaluate ambient differences in the chemical composition between the channels and among the experimental enrichment treatments, water samples were taken a few cm above the experimental units during the experimental runs using individual sterile 60 mL syringes, equipped with a 10 cm sampling tube. Right after sampling, water was filtered over GF/F filters and stored in 60 mL PE flasks at −20 °C until analyses. Samples were analysed at the laboratory of the chemical department of the Universidad Nacional del Sur (UNS). In spring, water samples were taken from three NDs of each treatment (AMB, N/P, N/P/Fe) within the SAO channel at 6 days in spring (14, 15, 18, 21 and 27 October and 1 November 2012). In addition, water samples were taken from the colonization set-up in the CONTROL channel (n = 1; 21, 27 October and 1 November) and analyzed for Si(OH)₄, PO₄³⁻ and NO²⁻ concentrations. In autumn, water samples were taken from three NDs of each treatment and channel at four days (16, 19, 22 and 26 March 2013) and analyzed for Si(OH)₄, PO₄³⁻, NO²⁻, NO³⁻, NH₄ and Fe II. In addition, total Fe was determined for all samples on 22 March and for the NPFE treatment on 26 March. A total of 129 water samples were investigated.

2.5. Laboratory Analyses

2.5.1. Composition and Biovolume of Macroalgal Recruits (GERMLING)

To determine the composition and biovolume of GERMLING, slides were scanned under a light microscope (Nikon Eclipse 80i) for 25 visual fields at 20× magnification (11.9 mm²). Given that early chlorophycean stages (e.g., Ulva spp.) appeared as colored cell aggregations intermixed within the diatom biofilm, we focused on the easy-to-distinguish and quantify rhodophyte germlings. Individual thallus length was measured, and biovolume was calculated according to thallus form, following [50]. The following five thalli forms were distinguished: (i) circle, (ii) ellipse, (iii) sphere, (iv) prolate spheroid, (v) cylinder and diameter, width and height or length measured.

2.5.2. Biomass Accumulation and Biochemical Composition of Phytobenthic Communities (MESO, MACRO)

To investigate the biomass accumulation of the whole benthic community under the different treatments, we calculated growth rates and standing stock for MESO and MACRO from dry weight (mg cm⁻²d⁻¹) during spring (SAO channel) and autumn (SAO and CONTROL channel). To investigate potential changes in algal chemistry and composition (e.g., marker pigments), pigment concentrations were determined for MESO and MACRO by extraction of methanol following the protocol of [51]. Chla (overall marker for photosynthetic activity), Chlb (marker for chlorophycean) and chlc1 + c2 (marker for ochrophyta) were calculated using the equations of [52]; pheopigments (as a marker for degraded photosynthetic products) and carotenoid 480 (as a marker for carotenoids) were calculated following [53]. In addition, to determine the assimilation of Fe in macroalgal tissue under the different treatments, subsamples from dried MACRO material were taken and Fe content was determined. Samples were analyzed at Laboratorio Centralizado de Química General y Análisis Elemental, CENPAT, CONICET (Protocol N°: 01929). The samples were mineralized by dry (400 °C), acid attacked (nitric pro-Merck analysis) to white ash and taken up in nitric acid (pro analysis, Merck, Darmstadt, Germany) (3% v/v) and hydrochloric acid (6% v/v) solution. The measurements were made using an Atomic Absorption Spectrophotometer (flame) IL 457 (Shimadzu Corperation, Kyota, Japan).

2.5.3. Composition of Macroalgal Communities (MACRO)

To study the taxonomic composition of MACRO in autumn, each tile was scanned at 3× magnification and six visual fields were counted with a stereomicroscope (Nikon SMZ 1500, Tokyo, Japan), equipped with an ocular grid (100 divisions) to allow quantitative measurement of individual taxa cover within each visual field. Microscopic analyses for MACROs were performed under the laboratory fume hood after the transfer and evaporation of formaldehyde-preserved material in filtrated seawater for 2 days.

2.5.4. Growth Rates, Carbohydrate and Total Carbon Content of ULVA

Growth rates (µa) were measured as changes in surface area (A) of Ulva cf. lactuca thalli, calculated as $\mu a=\left(lnAt-lnA0\right)t-1$, where A0 is the initial area and the final area after t days of incubation, following the protocol of [54]. To investigate potential changes in the carbohydrate concentration, subsamples of 10 mg lyophilized biomass of ULVA were analyzed following the methodology of DuBois et al. (1956) [55]. In this phenol–sulfuric acid-based approach, all present carbohydrates are reduced to glucose, whose concentration is determined photometrically. In addition, total carbon was calculated from the glucose as follows: $x\times {\displaystyle \frac{6\times y}{z}}=C (\mu {\displaystyle \frac{g}{mg}})$, where x = C₆H₁₂O₆ (Glucose), y = 6 × 12,011 u (atomic mass of Carbon) and z = 180,156 g/mol (molar mass of Glucose).

2.6. Statistical Analyses

Different non-parametric and parametric statistical analyses were applied to evaluate differences among the sites, treatments and seasons. Differences in the environmental parameters (water flow, water temperature, nutrient concentrations), as well as germling recruitment and biovolumes, growth rates, pigment concentrations, glucose and carbohydrate contents were calculated using analyses of variance (ANOVA), followed by Tukey’s test for significant differences. Data transformations (e.g., log (n + 1)) were conducted to meet the homogeneity of variances, as tested by Cochran’s prior analyses. In cases where no homogeneity was achieved, the non-parametric Kruskal–Wallis test was applied. The analyses were performed using Statistica v10 software (Statsoft, Tulsa, OK, USA). Non-parametric permutation analyses of variance (PERMANOVA) were applied to investigate differences in taxa structure and biomasses of phytobenthic units. For the taxonomic structure, Bray–Curtis similarity indices were calculated from percentage cover, prior to being transformed in order to scale down the importance of the highly abundant taxa. Identified differences between the factors were further investigated using the SIMPER methodology. All these non-parametric analyses data were conducted with the PRIMER v7 + PERMOVA software package (Primer-e, Auckland, NZ, USA). We tested for potential differences between (i) seasonality (factor season: spring, autumn), (ii) channels (factor channel: SAO and CONTROL), (iii) nutrient enrichment (factor nutrient: AMB, N/P, N/P/Fe) and grazer enclosure (open, caged, cage control). Data were visualized using Excel (Microsoft) GraphPrism version 9 (Graph Pad software, Boston, MA, USA).

3. Results

3.1. Physicochemical Characteristics of the Experiment

3.1.1. Natural Dynamics at the Experimental Sites

Differences were observed between the experimental channels at different seasons. The water temperature in both channels increased by about 3 degrees over the summer. Compared to the control channel, the SAO channel showed higher seasonal variations with the lowest temperatures in spring (13.0 °C) and the highest temperatures during late summer (16.5 °C) (

Table 2. Differences in the physicochemical properties between the two sampling sites in a tidal channel system at San Antonio Oeste (Northern Patagonia, Argentina) (SAO, CONTROL) studied in October 2012 (Exp 1; spring season) and March 2013 (Exp 2; late summer season). In EXP 1, temperature, pH and salinity were measured on 27 October and 1 November 2012 in both channels, using the multi-sensor device HORIBA U10 (Horiba, Kyoto, Japan). In EXP 2, water temperature was hourly logged from 15 February to 27 March 2013, using temperature loggers (Intec Electrónica, Buenos Aires, Argentina). Samples for nutrients were taken in spring (14, 15, 18, 21 and 27 October and 1 November 2012) and autumn (16, 19, 22 and 26 March 2013). Significantly higher values identified for either SAO or CONTROL channel in autumn are printed in bold and marked with *. s refers to single replicate evaluated during sampling period. Data based on ANOVA presented in Table A1 and Table A2.

Parameter	TEMP	pH	Salinity	PO4	NO2	NO3	NH4	SiOH4	Fe (II)	Fe tot
Unit	°C		ppt	µM	µM	µM	µM	µM	mgL−1	mgL−1
EXP 1: Spring season
SAO	13.0 ± 0.06	8.52 ± 0.06	35.4 ± 0.8	2.54 ± 1.12	1.31 ± 0.76	40.92 ± 31.04		30.12 ± 19.32
CONTROL	13.8 ± 0.01 *	8.54 ± 0.07	39.1 ± 0.2 *	3.10 ± 0.02	0.03 ± 0.04	0.38 s		21.52 ± 19.20
EXP 2: Late summer season
SAO	16.53 ± 2.46 *			6.86 ± 6.5 *	1.83 ± 0.74 *	28.35 ± 42.62 *	68.42 ± 52.03	41.14 ± 16.23	0.04 ± 0.03	0.09 ± 0.03
CONTROL	16.15 ± 3.11			1.58 ± 0.58	0.12 ± 0.04	3.64 ± 5.21	34.05 ± 34.3	51.19 ± 23.63 *	0.02 ± 0.02	0.15 ± 0.08

and

Table A1. Differences in the environmental conditions of the different locations. Environmental data showing differences in (a) water temperature and (b) water currents between experimental channels (SAO, CONTROL) and different exposure sites (experiment, colonization site) during Experiment 2 (EXP2: late summer). Water temperature was determined on 1 November 2012 during spring experiment (EXP1) and hourly measured during the autumn experiment (15 February to 27 March 2013). Water currents were calculated based on plaster of Paris (Doty 1971), exposed for three days in autumn (25–28 March 2013) at the colonization set-up and in additional NDs at both channels (n = 3). Data analyses are based on analyses of variance (ANOVA). Conclusion refers to the outcome of Levene’s test used as post hoc, indicating each significant difference (p < 0.05), indicated in bold print.

Factors	Df	MS	F	p
Water temperature
EXP 1: Spring
channels	1	7.21	95,956	<0.001
conclusion	SAO < CONTROL
Water temperature
EXP 2: Late summer
channels	1	46.346	5.883	0.015
conclusion	SAO > CONTROL
Salinity
EXP 1: Spring
channels	1	2.65 × 108	111,618	<0.001
conclusion	SAO < CONTROL
Water current
EXP 2: Late summer
channels	1	0.001	0.988	0.349
sites	1	0.000	0.115	0.743
channels × site	1	0.000	0.303	0.597
conclusion	No differences

).

Overall, the two channels strongly differed from each other in their chemistry, especially due to the high PO₄ load of the wastewater affected and a slightly less saline SAO channel (Table A1 and

Table A2. Differences in nutrient concentrations during the two experimental runs. Environmental data showing differences in ambient nutrient concentrations between the factors SITE (SAO, CONTROL), SEASON (EXP1: spring, EXP2: late summer), TIME (Day 2, 4, 7, 11) and NUT (AMB, N/P, N/P/Fe). Data analyses are based on analyses of variance (ANOVA) and non-parametric Kruskall–Wallis. Conclusion refers to the outcome of Levene’s test used as post hoc, providing each significant difference, indicated in bold. * = indicate log transformed data, ^H = indicate Kruskall–Wallis analyses. Conclusion refers to the outcome of Levene’s test, indicating each significant difference.

Nutrients		PO4			NO2			NO3			NH4			SiOH4			Fe(II)
Factor	Df	MS	F/H	P	MS	F/H	P	MS	F/H	P	MS	F/H	P	MS	F	P	MS	F/H	P
EXP1, EXP2: Ambient concentrations between seasons within the SAO channel
SEASON	1		7.57 H	0.006	1.995	3.48	0.07		2.53 H	0.11				874.37	2.65	0.115
Conclusion		EXP 2 > EXP 1
EXP1: Experimental conditions within site SAO
NUT	2		22.5 H	<0.001	0.01 *	0.13 *	0.87 *	0.12	1.07	0.37				38.8	0.12	0.89
Conclusion		AMB < N/P, N/P/Fe
EXP2: Ambient concentrations (EXP 2)
SITE	3		15.43 H	<0.001		17.29 H	<0.001	2.26 *	57.03 *	<0.001 *	7180	3.70	0.07	606.1	11.70	0.003	0.002	2.29	0.15
TIME	1		0.61 H	0.89		9.14 H	0.822	1.1 *	27.7 *	<0.001 *	3152	1.63	0.22	0.199 *	42.2 *	<0.001	0.0001	0.17	0.92
C × T	3							0.16 *	4.0 *	0.03 *	755.9	0.39	0.76	196.1	3.80	0.03	0.001	0.77	0.53
Conclusion		SAO > CONTROL			SAO > CONTROL			SAO > CONTROL; D2 > all; D4 > D11; SAO > CONTROL at D7 und D11						CONTROL > SAO; D2 > D4, D7 > D11; CONTROL > SAO at D7
EXP2: Experimental conditions within site CONTROL
NUT	2		25.5 H	<0.0001		23.9 H	<0.001	2.8	23.1	<0.0001		23.4 H	<0.001	42.9	0.11	0.89		23.6 H	<0.001
Conclusion		AMB < N/P, N/P/Fe			AMB, N/P < N/P/Fe			AMB < N/P, N/P/Fe		AMB < N/P, N/P/Fe							AMB, N + P < N + P + Fe
EXP2: Experimental conditions within site SAO
NUT	2	9.70 *	93.4 *	<0.000 *		10.33 H	0.006	51,482	22.4	<0.001	10.51 *	86.23 *	<0.001 *	846.9	1.94	0.16		23.56 H	<0.001
Conclusion		AMB < N/P, N/P/Fe			AMB < N/P, N/P/Fe			AMB < N/P, N/P/Fe		AMB < N/P, N/P/Fe							AMB, N + P < N + P + Fe

). The nutrient concentrations at the SAO channel showed a high seasonality, with PO₄ more than doubled in average concentrations from spring (2.5 ± 6.5 µM) to late summer (6.86 ± 1.5 µM) (Table 2, Figure A1).

As no difference was measured in spring for nutrient concentrations between the channels, PO₄, NO₂ and NO₃ showed significantly higher concentrations in the SAO channel in late summer (Table 2).

In both channels, SiOH₄ significantly decreased over time, whereas higher values were found in the CONTROL channel at both times (Table 2, Figure A1).

3.1.2. Measured Environmental Differences in the Experimental Set-Up

No difference was found in water velocity between the colonization and experimental sites (Table A1). Interestingly, a seasonal effect became visible in the nutrient concentrations measured above the experimental nutrient diffusers, depending on the nutrient identity, whereas overall higher values were measured in late summer (Figure 2). As no difference was measured for nitrogen (NO₂ and NO₃) in spring, PO⁴⁻ indicated a successful nutrient enrichment, by showing significantly (5 to 9 times) higher concentrations in the enriched N/P/Fe and N/P treatments. In contrast, at the end of summer, all enriched N, P and Fe sources were clearly reflected in the measured higher NO⁻³, NO⁻², NH⁴⁺, PO⁴⁻, FEtot and FEII values (Table A2, Figure 2).

3.2. Responses of Different Phytobenthic Assemblages to Top-Down and Bottom-Up Controls

A summary of the responses of the different phytobenthic units investigated is provided in

Table 3. Responses of different phytobenthic units (GERMLING, MESO, MACRO, ULVA) at two different sites (SAO, CONTROL), exposed to different factors: successional time (TIME; T1 = 3 days. T2 = 6 days); nutrient elevation (NUT; AMB, N + P, N + P + Fe, grazer exclosure (CAGE; open, caged, cage control) and seasonality (SEASON; SP-spring, LS-late summer). Responses are shown for germling numbers (number), taxa composition (TAXA), algal biomass growth (volume, growth, standing stock) and algal chemistry (CHEM: pigments/carbohydrates/Fe content); n.s. indicates non-significant differences identified in the statistical analyses presented in Table A3 and Table A4.

GERMLING
	Season	Site	Number	Taxa	Volume
TIME	SP	SAO	n.s.		T2 > T1
CAGE	SP	SAO	n.s.	T1: Cage control ≠ Caged	T2: open > caged
NUT	SP	SAO	T2: N/P/Fe > AMB	T2: N/P/Fe ≠ AMB	n.s.
MESO
	Season	Site	Growth	Pigments
SEASON	SP/LS	SAO	SP > LS
NUT	SP	SAO	N/P/Fe < Ambient
	LS	SAO	N/P/Fe < N/P	N/P/Fe > AMB for Chla, Chlc, carotenoids
	LS	CONTROL	n.s.	N/P/Fe, N/P > AMB for Chla, carotenoids N/P/Fe > AMB for Chlc
Site	LS	SAO/CONTROL	SAO > CONTROL	SAO > CONTROL for Chla, Chlc, carotenoids CONTROL > SAO for Chlb, Phaeo
MACRO
	Season	Site	Comp	Standing Stock	Pigments
SEASON	SP/LS	SAO		SP > LS
NUT	SP	SAO		n.s.
SITE	LS	SAO/CONTROL	CONTROL ≠ SAO	n.s.	CONTROL > SAO for Phaeo
NUT	LS	SAO	n.s.	n.s.
NUT	LS	CONTROL	n.s.	n.s.

, and details on differences in taxa composition are provided in

Table 4. Differences in taxa composition of GERMLINGs and MACRO communities based on PERMANOVA. GERMLING, showing differences in taxa composition based on recruitment numbers of communities grown in the SAO channel over different times (T1: 3 days, T2: 6 days) during spring season. Data showing results of three PERMANOVAs, investigating responses towards nutrient concentrations (NUT: AMB, NP, NPFE) and grazing treatment (CAGE: AMB, caged, cage control). MACRO community, showing differences between the communities exposed in the different channels (SAO, CONTROL) during late summer season. Data showing results of three PERMANOVAs, investigating responses of MACRO communities towards (a) experimental sites, testing differences between communities grown at the colonization set-ups in the different channels at different times (T0, T1); (b) response to nutrient enrichment, testing for differences between MACRO communities, exposed to different nutrient levels (AMB, NP, NPFE). Significant differences (p < 0.05) are marked in bold. Pair-wise tested based on 9999 Monte Carlo (MC) permutations are marked with *. Results of SIMPER analyses are given for each significant difference, showing percentage contribution of taxa to total dissimilarity between channels or times. Only taxa causing major differences (cut-off level 60%) are presented, and localization of higher taxa abundances is given in brackets.

GERMLINGS (EXP1: spring)
Factors	Df	MS	Pseudo-F	p	Pair-wise/SIMPER
(a) Ambient Conditions: Succession Time and Grazer Exclosure
TIME	1	7771.6	3.0535	0.0283	T1: Caged ≠ cage control [av.dis. 90%] 53% POLY (Caged) 27% CER (Caged) 20% INITIAL (Caged) HC: T1 ≠ T2 [av.dis. 92%] 74% POLY (S2) 16% ANO (S2)
CAGE	2	5290.1	2.0785	0.0543
TIME × CAGE	2	7989.5	3.1391	0.0131
Res	30
(b) Germlings (3 days): Cage and nutrients
CAGE	2	9345.9	3.0941	0.016	Caged ≠ HC [av.diss: 92%] 51% POLY (Caged) 39% INITIAL (Caged) 11% CER (Caged)
NUT	2	5726.5	1.8958	0.1016
CAGE × NUT	2	3443.8	1.1401	0.3397
Res	35	3020.6
(c) Germlings (6 days): Cage and Nutrients
CAGE	2	3911	1.8748	0.07	N/P/Fe ≠ AMB [av.dis. 69%] 32% POLY (N/P/Fe) 26% INITIAL (N/P/Fe) 23% CER (N/P/Fe)
NUT	2	4597.3	2.2038	0.037
CAGE × NUT	2	2024.2	0.97032	0.449
Res	35
MACRO taxa composition (EXP2: late summer)
Factors	Df	MS	Pseudo-F	p	Pair-wise/SIMPER
(a) Differences between the experimental sites during late summer (colonization set-up)
SITE	1	2839.8	9.96	0.002	T0: CONTROL ≠ SAO * [av.diss.:56%] 21% Ulva germlings (SAO) 14% Polysiphonia (CONTROL) 14% Turbular Ulva (CONTROL) 13% Tube-dwelling diatoms (CONTROL) 10% Chaetomorpha (SAO) 7% Cladophora (SAO) T1: CONTROL = SAO *
TIME	1	1312.5	4.60	0.01
SITE × TIME	1	2189.1	7.67	0.003
Res	8
(b) Response to nutrient enrichment and sites (NDs)
SITE	1	12,249	28.08	0.001	CONTROL ≠ SAO * [av.diss.:49%] 17% Tube-dwelling diatoms (CONTROL) 15% Ulva germlings (SAO) 14% Polysiphonia (CONTROL) 12% Foliose Ulva (SAO) 10% Turbular Ulva (CONTROL)
NUT	2	381	0.87	0.55
SITE × NUT	2	720	1.65	0.14
Res	24

. Details for each phytobenthic unit are given below.

3.2.1. Response of Rhodophyte Germlings (GERMLINGs)

In spring, a total of five different rhodophyte germling types, mainly composed by members of the Ceramiales, were observed after 3 days of exposure, comprising: (i) sphere shaped tetraspores (TETRA), (ii) prolate spheroid shaped initial stages, mainly members of Ceramiales, (iii) prolate spheroid shaped germlings of Ceramium (CER), (iv) cylindrical shaped Polysiphonia germlings (POLY) and (v) cylindrical shaped Anotrichum like germlings (ANO), whereas additional two taxa appeared after 6 days: (vi) crustose stages (CRUST; e.g., Hydrolithon sp.) and (vii) foliose like Porphyra (POR) (Figure 3).

Over the initial 6 days, germlings increased their thalli lengths from 34 ± 13 µm after 3 days to 195 ± 105 µm after 6 days, leading to a significant 5-fold increase in their biovolume from 0.9 × 105 ± 0.4 × 105 µm³ to 4.5 × 105 ± 8.7 × 105 µm³ (Figure 3, Table 3 and Table A3), whereas the recruitment numbers strongly varied from 8 ± 5 individuals/cm² to 24 ± 29 individuals/cm² but did not significanty differ over the initial days (Figure 3, Table 3 and Table A3).

The grazer control treatment (cage) strongly impacted the germlings, with about 50% lower biovolume measured after 6 days (Figure 3, Table 3 and Table A3), without changing the taxonomic composition (Table 4), or the recruit numbers, which stayed with an average of 17 individuals/cm² stable over time (Figure 3, Table 3 and Table A3). The assemblage structure in the cage control differed temporarily from that at the exclusion treatment after 3 days (Table 4), mainly caused by the small numbers of Ceramiales recorded in the cage control treatment, which increased after 6 days.

Iron fertilization (N/P/Fe) significantly increased the germling numbers after six days to 63 ± 26 individuals/cm² compared to 24 ± 29 individuals/cm² measured under ambient conditions (Figure 3, Table 3). Elevated numbers of Polysiphonia, Ceramium and unidentified initial stages in the N/P/Fe treatment significantly changed the taxa composition (Table 3 and Table 4). Despite no significant effect being found, maximum thallus lengths were repeatedly found in Polysiphonia grown in the open N/P-enriched treatments, reaching 275 µm and 450 µm after 3 and 6 days, respectively.

3.2.2. Responses of Mesoalgal Assemblages/Units (MESO)

The phytobenthic assemblages that had settled and grown for about a fortnight under the different conditions were mainly composed of different diatoms, intermixed with crustose and filamentous macroalgae, forming a low (a few cm high) tuft-like assemblage. A significant four-fold higher biomass accumulation was observed in the MESO assemblages during spring (3.8 ± 2.1 mg × cm⁻²d⁻¹) compared to autumn (0.9 ± 0.4 mg × cm⁻²d⁻¹) in the SAO channel (Table 3 and

Table A4. Differences in growth rates of MESO and standing stock of MACRO in response to different factors, including: CHANNEL (SAO vs. CONTROL), SEASON (spring, summer) and NUT (AMB, N/P, N/P/Fe), based on analyses of variances (ANOVA). Conclusion refers to the outcome of Levene’s test, indicating each significant difference indicated in bold.

		MESO Growth Rates			MACRO Stock
Factor	Df	MS	F	p	MS	F	p
(a) SEASONS in SAO channel
NUT	2	0.102	5.582	0.008	0.05	0.752	0.482
SEASON	1	0.698	24.403	<0.001	8.85	144.253	<0.000
N × S	2	0.026	1.440	0.251	0.02	0.320	0.729
Conclusion		AMB > N/P/Fe Spring > summer			Spring > summer
(b) CHANNELs in EXP2
CHANNEL	1	0.788	24,416	<0.000	0.284	3.930	0.058
NUT	2	0.024	0.734	0.490	0.048	0.663	0.524
C × N	2	0.114	3546	0.045	0.014	0.201	0.819
Conclusion		SAO > CONTROL CONTROL: n.s.; SAO: N/P > N/P/FE;

, Figure 4). Differences between the channels were prominent, as a three-fold lower biomass accumulation was measured in the CONTROL channel (0.3 ± 0.1 mg × cm⁻²d⁻¹) (Table 3 and Table A4, Figure 4). The difference between the channels was also reflected in the pigment concentrations (Table 3 and

Table A5. Differences in pigment concentrations of MESO and MACRO in response to different factors, including: CHANNEL (SAO vs. CONTROL) and NUT (AMB, N/P, N/P/Fe), based on analyses of variances (ANOVA) and Kruskall–Wallis, indicated by (H). Significant differences are printed in bold. Conclusion refers to the outcome of Levene’s test, indicating each significant difference.

(III) Pigment concentrations in MESO and MACRO communities.
		chla			chlc			chlb			carotenoids			phaeo
Factor	Df	MS	F/H	P	MS	F/H	P	MS	F/H	P	MS	F/H	P	MS	F/H	P
MESO
CHANNEL	1	2.91	232.6	<0.000	2.68	234.59	<0.000	0.81	12.5	0.002	2.72	288.4	<0.000	7.43	36.8	<0.000
NUT	2	0.14	11	<0.000	0.06	5.57	0.01	0.05	0.77	0.473	0.1	11.09	0	0.19	0.93	0.409
C × N	2	0.07	5.27	0.013	0.03	2.5	0.103	0.05	0.77	0.473	0.05	5.39	0.012	0.19	0.93	0.409
Conclusion		SAO > CONTROL CONTROL: AMB < N/P, N/P/Fe SAO: AMB < N/P/FE			SAO > CONTROL N/P/FE > AMB			CONTROL > SAO			SAO > CONTROL CONTROL: AMB < N/P, N/P/Fe SAO: AMB < N/P/FE			CONTROL > SAO
MACRO
CHANNEL	1	0.56	2.56	0.123	0.006	1.12	0.3	1.5	1.08	0.31	0.02	1.12	0.3		22.19 (H)	<0.000
NUT	2	0.06	0.29	0.749	0	0.01	0.988	1.49	1.07	0.36	0	0.38	0.69		0.55 (H)	0.756
C × N	2	0.64	2.93	0.072	0.011	2.04	0.151	1.36	0.97	0.39	0.06	2.94	0.07
Conclusion														CONTROL > SAO

, Figure 4). The growing MESO communities significantly differed in their pigment concentrations, with higher concentrations of chlorophyll a and carotenoids, indicating an overall higher amount of photosynthetic activity in the SAO channel and a higher amount of degraded biomass and a higher relative portion of Chlorophycean in the CONTROL channel (Table 3, Figure 4). For the MESO assemblages, the N/P/Fe treatment had a significant effect, but the direction and strength of the phytobenthic responses depended on the site and season.

The growth of the CONTROL assemblage increased with nutrient addition, whereas the growth rates of the SAO assemblage decreased significantly in the N/P/Fe treatment during spring and were lower than in the N/P treatment in AUTUMN (Table A3, Figure 4). Also, pigment concentrations measured in AUTUMN responded to the altered nutrient levels in different ways. In the CONTROL channel, a nutrient-driven increase in Chla, Chlc and carotenoids was observed, while no difference between the ambient and N/P treatment was observed in the SAO channel (Table 3, Figure 4).

3.2.3. Responses in Macroalgal Assemblages (MACRO)

Also, the macroalgal assemblages showed seasonal variations, as observed in their significantly higher standing stock in spring (EXP1) measured in the SAO channel (Table 3, Figure 4). Differences between the channels became visible in the taxa composition analyzed in late summer (EXP2). A total of 11 taxa were distinguished and quantified, including one Phaeophyceae (Ectocarpales), three Rhodophytes (Polysiphonia, Ceramium, Anotrichium), five Chlorophytes: foliose Ulva (here named Ulva cf. lactuca), turbulose Ulva, Ulva germlings, Cladophora, Chaetomorpha, and one taxon of macroscopic growing Bacillariophyta (tube-dwelling diatom), partly identified as Berkeleya rutilans. In addition, one faunal component, “peracaridean tubes”, mainly built by amphipods, identified as Monocorophium insidiosum, was included. Similar to the MESO communities, MACRO assemblages had over ten times significantly higher standing stock during the spring season measured in the SAO channel (Table 3, Figure 4).

The MACRO assemblages differed in their structure between the channels at the beginning of EXP2 (late summer), as the SAO assemblages were mainly composed of the Chlorophytes ULVA germlings, foliose Ulva, Chaetomorpha and Cladophora, and the CONTROL community was composed of a mixture of tube-dwelling diatoms, Polysiphonia and turbulose ULVA (Table 4, Figure A2). The differences in taxa composition vanished at the end of the experiment in the colonization set-up, whereas they persisted in the exposed communities in the NDs.

Under additional nutrient supply, no difference was observed even in the standing crop, despite an average three times higher values (425 ± 800 mg/cm²) being found for the standing stock grown in the N/P-enriched treatment in the SAO channel during the spring season, mainly due to measured high variability (Figure 4). Also, the additional iron enrichment did not significantly increase the macroalgal growth, and no significant differences were found in the MACRO biomass, which showed an average iron content of 6.3 ± 1.8 mg/gDM in the MACRO of the CONTROL and about 7.1 ± 1.2 mg/gDM in the SAO channel. For the pigments, a significantly higher concentration in phaeopigments was measured in the CONTROL assemblages at the end of Exp 2 (Table A5, Figure 5).

3.2.4. Responses in ULVA

An average growth rate of 0.13 ± 0.02 µa and concentrations of 12.9 ± 2.7 µg/mg carbohydrates and of 5.2 ± 1.1 µg/mg of total carbon were observed for ULVA exposed to their natural conditions in the SAO channel (

Table 5. Difference in chemical macroalgal tissue composition of MACRO and ULVA units. exposed to different treatments (AMB, N/P and N/P/Fe) during late summer (EXP2). Data showing averages ± S.D.

	AMB	N/P	N/P/Fe
MACRO: Fe (mgg−1 DW)
SAO	7.11 ± 1.25	5.86 ± 2.29	8.55 ± 1.85
CONTROL	6.34 ± 1.80	7.16 ± 7.16	7.20 ± 1.37
ULVA: Carbohydrates (µgmg−1 DW)
SAO	12.9 ± 2.7	16.1 ± 8.9	17.0 ± 6.9
CONTROL	8.7 ± 1.3	9.8 ± 4.3	12.7 ± 2.5
ULVA: Total carbon (µgmg−1 DW)
SAO	5.2 ± 1.1	6.4 ± 3.5	6.8 ± 2.8
CONTROL	3.5 ± 0.5	3.9 ± 1.7	5.1 ± 0.4

). Comparing the different sites, no difference was found in the growth or total carbon, but for the amount of carbohydrates, which were significantly elevated in the SAO channel (Table 3). Additional nutrient supply did not affect the ULVA, neither in growth nor in chemistry (carbohydrates or total carbon) (Table 3).

4. Discussion

Our results confirm the initial hypothesis, demonstrating that a nutrient pulse at different successional stages led to distinct phytobenthic assemblages. This outcome was influenced by a combination of nutrient composition, grazer abundance and site identity. The most pronounced responses were observed during the earlier successional stages. Below, we discuss the main findings and explore the underlying mechanisms that may explain these patterns. Seasonal differences were not only reflected in decreasing water temperature but also in nutrient concentrations. Ambient PO₄ levels strongly increased in the SAO channel during late summer, which might partly be explainable by decreasing nutrient uptake activities of the declining algal stock at the end of the growth season. Differences in the nutrient load between the channels were more prominent in autumn, characterized by elevated PO₄⁻, NO⁻³ and NO⁻² levels in the polluted SAO channel. The measured ambient DIN concentrations during this experiment (of about 98.8 ± 55.6 µM) exceeded the recorded values of other known eutrophied coastal systems, like the Venice Lagoon (11.6 µM DIN in 2011) [56] or the German North Sea coast (up to 85 µM) [57]. Interestingly, despite the above enriched treatments, these values even peaked up to 3138 ± 2516 µM (NP treatment in the SAO channel), phytobenthic growth and development were supported over experimental time, and no detrimental effect was observed. Nevertheless, strong differences were found in phytobenthic assemblages between channels and seasons, whereas the strongest responses were observed at early successional stages (GERMLINGS and MESO). The algal assemblages present in the tidal channels provide high capabilities to adapt to excess nutrient loads, responding with increased germling recruitment, elevated pigment concentrations and increased growth rates. Interestingly, as observed during the present study, responses to altered nutrient levels strongly decreased with successional stage, indicating a high adaptive potential of the present taxa.

Marine coastal eutrophication in Patagonia is driven by anthropogenic nutrient enrichment from watershed inputs [58] and aquaculture [59]. Opportunistic algal proliferation and shifts in benthic communities have been reported in different areas of Argentina and Chile, supporting the regional relevance of our findings. In northern Argentine Patagonia, δ¹³C and δ¹⁵N isotopic signatures demonstrate terrestrial nitrogen flows penetrating benthic food webs, indicating altered primary sources and trophic structure from coastal nutrient loading [15]. Along Argentina’s Nuevo Gulf, macroalgal assemblages exhibit eutrophication symptoms near urban sewage outfalls, including reduced diversity and shifts in biomass composition [60]. In Chile, nutrient release from intensive salmon farming has been implicated in localized “dead zones” below pens and correlated with red tide events, such as the 2016 Chiloé crisis exacerbated by organic waste dumping [59].

The observed high phytobenthic and potentially also planktonic activity in the SAO channel might also explain the observed decline in ambient SiOH₄, a key molecule especially for diatoms. In fact, blooms of tube-dwelling diatoms were observed in the SAO channel during the study time [18], and measured concentrations of the Ochrophyta marker pigment Chlorophyll c seem to support their high abundances within the MESO assemblages. Overall, the studied phytobenthos was mainly composed of different genera known as fast nutrient responders, like the diatom Berkeleya [18] or the green macroalga Ulva [12], able to quickly take advantage of nutrient pulses. In addition, the observed differences in red algal germlings indicate a quick response in fertility of the Ceramiales, especially of the dominant genus Polysiphonia. Given that invasive species usually occur in disturbed, high-resource environments [61,62], our observations may correspond to the presence of invasive Polysiphonia forms, which are known to be present along the Patagonian coastline [63,64,65]. Overall, the extreme environmental conditions found in the tidal channels of the San Antonio Bay, characterized by tide-driven drastic changes in water level, connected with strong alterations in light, chemistry and anthropogenic pressure, support a highly adaptive biota able to tolerate such daily natural alterations, and deal with the increasing anthropogenic pressures, measurable in high nutrient influxes.

The observable highest effects were not always observable in the highest Fe-enriched treatment (N/P/Fe). The phytobenthic responses seem to depend not only on the successional stage of the assemblage but also on the nutrient history (e.g., accumulation of excess nutrients) of the environment, as indicated by different responses observed in the two channels at different seasons. Playing a key role in the chlorophyll and chloroplast biosynthesis, the micronutrient Fe is considered to increase the biomass of photosynthetic organisms [66]. To overcome the natural low iron availability in marine waters, algae evolved different strategies, including the reduction and restructuring of the photosynthesis apparatus, as well as the storage of iron due to special proteins (ferritins), as observed for pennate diatoms [23] and macroalgae like Ulva [67]. Also, in our study, we observed a positive effect of additive iron on the macroalgae, measured in a significant increase in rhodophyte germling numbers and a slight but irregular elevation (on average, about 20%) in incorporated iron concentration in thalli of Ulva cf lactuca.

Given the observed variability of local conditions and biological responses, the definition of nutrient thresholds is difficult. Our findings suggest that increases in PO₄ and DIN concentrations, especially NO₃⁻ and NH₄⁺, above certain levels (e.g., >6 µM PO₄ and >60 µM DIN in the SAO channel) led to marked shifts in early-stage communities. These include increased rhodophyte germination density and higher chlorophyll–carotenoid content in mesoalgal assemblages. Such responses point to biologically meaningful thresholds where community composition and function begin to change, reinforcing observations from previous studies on macroalgal blooms under eutrophic conditions [12,68].

Overall, the system under study seems to have reached its natural nutrient uptake limits, as observed in the growth inhibition of mesoalgae exposed to additional Fe, within the already nutrient-loaded SAO channel. But it remains questionable if the observed decline in biomass corresponded to physiological restrictions of the exposed taxa. In fact, elevated chlorophyll (Chla and Chl c) and carotenoid levels in the MESO assemblages in the iron-enriched treatments seem to contradict this hypothesis.

Consequently, other interacting factors might be considered to be responsible for the observed phytobenthic patterns (e.g., an increase in germling numbers, elevation of photosynthetic pigments, but partly even decreased growth). The high grazing pressure observed in former studies [32,36] and partly addressed in this work (with the cage exclusion experiment) might provide a potential explanation. Coupled to the observed seasonal differences, a high grazing pressure might also explain the observed high variance in Ulva growth rates. Nonetheless, the herbivorous community at the experimental sites was shown to adapt its consumption in dependence on food quality. As shown in an experimental study by [36], Ulva cf. lactuca was preferably grazed at low N tissue concentrations, which was achieved by PO₄³⁻ fertilization, whereas additional NO₃⁻ did not increase the grazer activity. In addition, reported site-dependent differences in the presence of algal grazers, e.g., chitons (Chaetopleura isabellei), keyhole limpets (Fissurella spp.), amphipods and smaller snails (Tegula spp.), showing higher abundances in the SAO channel [32,36] might play an important role.

Contrary to other studies [31], the presence of cages did not favor algal growth; moreover, a decrease in the biovolume of the GERMLING assemblages was observed regardless of their nutrient enrichment level. Given the mesh size of 1 cm and the observed high abundance of smaller mesofauna (e.g., peracaridean tubes on the settlement tiles), it is highly probable that the exclusion worked for predators rather than for grazers, thus providing shelter for smaller grazers. In a former study in San Antonio Bay, an increase of up to 60% in Ulva sp. biomass accumulation was found when grazers were excluded, using a 1 mm mesh [36]. Even when larger grazers have been described in the area [32,36], the results of the present study suggest that the highest herbivory pressure is given by small grazers. Moreover, a food web study carried out in San Antonio Bay also indicates that macroalgae do not represent a main contribution to larger fauna (Becherucci et al. 2019 [15]). Especially smaller crustaceans like amphipods or isopods are known to graze on a variety of benthic diatoms [69] as well as on different types of macroalgae. In this regard, it might also be possible that the observed discrepancy in the iron-enriched MESO assemblages (increasing pigments but decreasing biomass with Fe addition) is directly linked with a change in grazer behaviour as the algae might become more palatable due to their altered chemistry [36].

Overall, as we observed a lack of response of MACRO assemblages and ULVA under nutrient enrichment contrasts, we suspect that the macroalgal assemblage has already reached its maximum growth under the given conditions. But the relative contribution of different factors, such as grazing pressure, substrate quality and nutrients, to shape the observed assemblages remains unclear. Given the high observed environmental dynamics and variety in factors present, it is thinkable that further top-down, bottom-up relations were overlooked. Also, further insight into the physiological boundaries of apparent key taxa, like Ulva, Polysiphonia, Berkeleya or Monocorophium would help to better understand the existing patterns and potential consequences of further pollution of adjacent or comparative coastal areas.

5. Conclusions

Overall, this study provides an insight into the different phytobenthic strategies coping with rising coastal eutrophication. The results of the present study highlight the importance of the successional stage at the time of the nutrient pulse, as well as the importance of the presence of mesograzers and the nutrient history of the environment. As the human population continues to grow, particularly in coastal areas, the frequency of nutrient pulses is expected to increase. These human-driven changes compound other stressors, such as temperature increases, pH shifts and the spread of invasive species. Understanding how these factors interact and transform coastal ecosystems is a major challenge for contemporary ecological research.