Metabolic Rates of Rainbow Trout Eggs in Reconstructed Salmonid Egg Pockets

Abstract

In situ evaluations of the metabolic rates (i.e., respiration and excretion) of salmonid eggs are mostly indirect, focusing on the sampling of hyporheic water from wild or artificial nests. Comparatively, experimental studies carried out under controlled, laboratory conditions are less abundant due to methodological difficulties. This study presents a novel experimental setup aimed to address this issue and enable the measurement of oxygen and dissolved inorganic nitrogen fluxes in simulated rainbow trout (O. mykiss) egg pockets. The experimental setup consists of reconstructed egg pockets in cylindrical cores under flow-through conditions. Live and dead eyed-stage eggs were incubated in a natural, sterilised gravel substrate. Hyporheic water circulation was ensured using peristaltic pumps, with the possibility to collect and analyse inflowing and outflowing water for chemical analyses. Microcosm incubations, with closed respirometry of eggs in water alone, were also carried out in order to infer the importance of microbial respiration in the simulated egg pockets. The results show an increasing trend in oxygen demand, due to the development of biofilm in the reconstructed egg pockets and increased egg respiration rates. Moreover, egg pockets showed positive ammonium net fluxes connected with the advancing developmental egg stage, while nitrate removal peaked during the last phase of the experiment, mainly due to the formation of oxic-hypoxic interfaces, leading to couple nitrification–denitrification processes. The suggested approach enables to test a number of in situ situations, including the effects of extreme hydrological conditions, sediment clogging and sudden changes in water chemistry or temperature on the survival and metabolic performances of nests, at different egg development stages.

1. Introduction

Salmonids lay their eggs within river bottoms up to 10–30 centimetres below the interface between the water and the gravel substrate [1]. The nest, called also redd, is formed by several egg pockets that represent metabolic hot-spots within an inorganic matrix with flowing hyporheic water. While preparing nests before egg deposition, salmonids displace gravel and finer grained sediments in order to remove fine material, which is exported downstream by currents [2]. This mechanical action has a multifold aim: it improves water circulation within the nest, and it removes organic fractions potentially fuelling microbial activity and associated oxygen (O₂) consumption and metabolic end product accumulation.

Within egg pockets, eggs respiration and excretion rates and their mortality can locally modify the water chemistry [3]. The extent of such changes depends on water renewal time and may result in positive or negative feedback for mortality [4]. In the context of climate change, hydrological extremes and to improve the efficacy of river restoration actions targeting the recovery of salmonid populations, it is important to focus research activities on salmonid reproduction areas and specifically on the fine dynamics occurring in spawning sites during eggs development [3,4,5]. Thus, a large body of literature has explored these issues to understand the dynamics of eggs development and of the factors that regulate eggs mortality [2,6,7,8]. Pivotal is the role played by intragravel water circulation related to the maintenance of low temperature ensuring high O₂ solubility [9]. These factors, in turn, are interconnected with the salmonid eggs metabolic rates, that, like other organisms [10], are expected to increase with the rising temperatures under the ongoing climate change scenario [11]. Because the O₂ supply to the eggs is dictated by a diffusion mechanism, those salmonids species producing smaller eggs, and thus with a larger surface-to-volume ratio, will be advantaged since their lower metabolic rates will translate to an higher thermal tolerance [12].

The most recent studies assessing the intragravel conditions inside salmonid nests have been generally carried out in situ by means of a wide range of pore water samplers, egg incubation boxes and substrate collectors [3,4,8,13]. Overall, such studies try to evaluate changes in water chemistry and infer underlying processes or water circulation-related issues as the dominance of upwelling or downwelling conditions. Nonetheless, in situ assessments often lack in spatio-temporal precision when they evaluate dissolved oxygen (DO) content and egg survival rates [14]. This is mainly because of the steep variations in the DO concentrations due to variable flow regimes, the uneven gravel composition in the redd and the difficulties in locating the egg pocket [13,15]. Additionally, on field studies focussing on sedimentary structure as a metric to define survival rates miss to catch the influence of the biochemical oxygen demand (BOD) and surface–groundwater (sw-gw) interactions as additional drivers for DO availability [4,13]. Thus, appraisal accounting solely for the DO concentration can fail in detecting zones of DO sags due to stagnation linked with low intragravel flow velocities [3,6,8].

In recent times efforts have been made to disentangle the complex redd structure to assess the related flowpaths and DO distribution. Tonina and Buffington [16], for example, developed a three-dimensional hydraulic model to simulate the influence on hyporheic circulation into a pool-riffle sequence after the redd construction. The same authors found that the fine fraction is winnowed by the female out of the nest, remarkably altering the hydraulic conductivity, leading to a cascade effect on hyporheic flowpaths distribution as well as on O₂ circulation. High-scale resolutions of intragravel patterns at different flow velocities and detailed observations of DO transport were conducted by Cardenas [17], who employed a recirculating flume where an artificial salmon redd was built. Nevertheless, despite the high resolution conferred using computational models [16] as the DO mapping [17] inside reconstructed nests, to our knowledge there are no studies evaluating the respiratory and excretion rates of a typical salmonid egg pocket within the gravel matrix.

In this study fluxes of dissolved O₂ and inorganic nitrogen (ammonium—NH₄⁺ and nitrate—NO₃⁻) were studied in two sets of reconstructed egg pockets under controlled laboratory conditions. One set, in triplicate, included 100% alive eyed-stage eggs, whereas another set, in triplicate, contained 50% alive and 50% dead eggs. The egg pockets were reconstructed in plexiglass transparent cylinders packed with a mixed inorganic substrate composed of sand and gravel and eggs. Cylinders were provided with a water circulation system, ensuring constant flow of stable quality, temperature and O₂ saturated water, allowing for the measurement of inflowing and outflowing water chemistry and flux calculations. In order to quantify the contribution of eggs to the whole system respiration, eggs were also incubated in water alone. This study has a twofold aim: (1) to present a novel methodological approach that can be used to analyse whole egg pocket metabolic rates under ideal or pristine flows and water chemistry conditions and under a wide range of simulated conditions that mimic ongoing, climate-related changes in water flow, direction, temperature and chemistry; and (2) to present the results of a test incubation were two treatments (100% alive vs. 50% alive and 50% dead eggs) were contrasted.

2. Methodology

2.1. Microcosm Setup

A batch of eggs were kept in the trays of a vertical incubator and used to run closed respirometry incubations in order to quantify the oxygen (O₂) demand and N-NH₄⁺ excretion from single individuals from the three main developmental stages (eyed, nearly hatched, yolk sack absorbed). Respiration and excretion rates were measured by means of 22 mL glass chambers equipped with an opdote sensor spot [18] for continuous DO measurement (FireStingO2, PyroScience GmbH, Aachen, Germany) using the following formula:

$$RespirationorExcretion\left(\mathrm{m}\mathrm{g}\mathrm{or}\mu \mathrm{g}{\mathrm{e}\mathrm{g}\mathrm{g}}^{-1}{\mathrm{h}}^{-1}\right)=\frac{∆C\times V}{n\times ∆t}$$

where ΔC/Δt (mg or µg L⁻¹h⁻¹) is the variation of the DO or N-NH₄⁺ concentration during the incubation, V (L) is the volume of the incubation chamber, and n is the number of incubated eggs.

Chambers were equipped with a stirring device in order to keep water moving to avoid O₂ sags and stratification. Data were pooled from glass chambers in which each stage was incubated at four different densities (n = 5, 10, 15, 20 per chamber) to account for any density-dependent mechanism changing the DO consumption or the N-NH₄⁺ excretion. Because of their larger size, final stage alevins fit in the chambers only at 1, 3 and 5 individuals at a time. At time zero the water in the chambers had the same temperature and O₂ saturation of the mesocosms in which egg pockets were reconstructed (see next paragraph). The incubation started with the chambers were closed, and fluxes were measured in dark conditions. Also, each batch had one control chamber with only water, to correct eggs respiration and excretion rates and to account for any change in the target parameters. In order to obtain results describing metabolic rates under optimal conditions, the experiments ended when the DO concentration reached 8–9 mg L⁻¹, from an initial DO concentration of 11.760 ± 0.003 mg L⁻¹. The incubation time was set to avoid the critical level of 7.2 mg L⁻¹ defined by Alderdice [19] for O. keta that would affect the respiration rate. The safety DO concentration is defined by that threshold above which the respiratory rate is independent from the external DO concentration, thus keeping growth and metabolism rates unmodified [19]. At the end of the experiment, a water sample was collected from each microcosm and filtered (Frisenette GF/F filters) into plastic test tubes to account for the N-NH₄⁺ concentration using standard colorimetric methods [20] and thus obtain individual excretion rates.

2.2. Mesocosm Setup

The experiment was carried out in a recirculating aquaculture system (RAS) at the Fisheries and Aquaculture Laboratory of Marine Research Institute, Klaipeda University (Lithuania). Rainbow trout (O. mykiss) eyed-stage eggs at 270 degree days (dd) were supplied by Dabie hatchery, Poland on 19 April 2023. Subsamples of the eggs were distributed in three control trays with 100 eggs each to assess the natural mortality of the strain and to track the developmental stage. In the meanwhile, 6 reconstructed trout egg pockets were built by packing spawning ground (pooled gravel and sand and silt) into 6 transparent plexiglass cylinders (i.e., cores, height 40 cm, inner diameter 8 cm). To comply with the security standards of the RAS plant, the substrate was initially sterilised with oxygen peroxide (concentration 30%). Once the cylinders were filled with water, the substrate and the eggs were gently transferred into the cylinders, and egg pockets were reconstructed by pooling bigger stones together and then laying the eggs on top by means of a plastic spoon in order to ensure an even distribution as much as possible (Figure 1). Three cylinders were packed with 100 alive eggs each, hereinafter called “L” (live); whereas three cylinders were packed with 50 alive and 50 dead eggs, adequately mixed, hereinafter labelled as “L + D” (live and dead), in order to simulate the processes occurring in a partly compromised environment with high rates of mortality. In each cylinder, the eggs were distributed in a ~25 cm column. The bottom lids of each cylinder, consisting of rubber bungs, were drilled and connected to 5 silicone tubes, ensuring a constant and similar one-way water flow (from the bottom to the top) inside the reconstructed egg pockets. The upper end of each cylinder was sealed with another rubber bung provided with a single hole and a pipe collecting the overflow. The water circulating in the six packed mesocosms was ensured using peristaltic pumps. All cylinders were submersed in a large aquarium, ensuring constant temperature (Figure 2). The outflow pipe of each cylinder discharged continuously the water into a 50 mL falcon tube fixed on the outer cylinder wall and ensuring the possibility to subsample the water. The peristaltic pump velocity was adjusted in order to provide a constant outflow of 187 mL min⁻¹. Pilot tests were previously carried out using dye as a tracer flowing into the same mixed substrate in order to reproduce a water velocity in the porous medium of 1440 ± 60 cm h⁻¹ (average ± standard deviation). We confidently set this velocity in order to guarantee intragravel survival at the ranges of DO concentration supplied during the experiment (11.760 ± 0.003 mg L⁻¹). Such velocity is far above the optimal range of 15–500 cm h⁻¹ defined by Greig [4] in UK rivers to guarantee optimal egg survival in Atlantic salmon (S. salar) nests. Such range was additionally supported by Nika [21], who found high rates of survival with a velocity range of 1000–10,000 cm h⁻¹ for the local sea trout populations in nests with a similar substrate as the one used here. After two days of acclimatisation in the cores (eggs at 286 dd), periodical DO measurements were started by means of an opdote sensor needle (FireStingO2, PyroScience GmbH) inserted in the falcon receiving the outflowing water. Besides O₂, N-NH₄⁺ and N-NO₃⁻ were also measured via water sampling from the falcon and spectrophotometry. The DO demand and inorganic nitrogen fluxes were expressed on a per core basis (i.e., single core) and related to the main stage of development (i.e., “eyed egg”, “alevin”), as recorded in the nearby trays, with the following formula:

$$\mathrm{F}\mathrm{l}\mathrm{u}\mathrm{x}\mathrm{e}\mathrm{s}\left(\mathrm{m}\mathrm{g}\mathrm{o}\mathrm{r}\mathrm{\mu }\mathrm{g}{\mathrm{L}}^{-1}{core}^{-1}\right)=(C_{out}-C_{in})\times Q$$

where C_out and C_in (mg or µg L⁻¹) are the concentrations of the DO or dissolved inorganic N at the core outlet and inlet, respectively, and Q (L h⁻¹) is the water flow.

Following the previous definition [19], we use the term “embryo” to point out an unhatched fish, still inside the egg, while the term “larva” refers to a hatched fish, namely “alevin,” with its yolk sack. Throughout the duration of the experiment, the gravel cores were always in the dark to avoid algal growth, and the RAS supplied water at the constant temperature of 8^◦C and 100% DO saturation. The experiment ended on 7 May, when, for reference, the vitelline sack was completely adsorbed into the control trays. Then, the cylinders were opened and unpacked, and the juveniles were counted to assess the survival mortality. The substrate was analysed to detect the percentage of fines (<2 mm and <0.063 mm) by means of different sieve diameters following the methodology of Nika [21]. Once cores were opened to remove and count the alevins, three cores (control) were prepared with a similar approach and re-incubated for one additional day to account only for the flux rates of the bare intragravel environment.

2.3. Data Analysis

Respirometric measurements conducted in closed chambers were tested for differences in the DO demand (mg O₂ egg h⁻¹) and ammonium excretion (µg N-NH₄⁺ egg⁻¹ h⁻¹) among the three stages using the non-parametric Kruskal–Wallis test due to the absence of homoscedasticity and normality in the data. With significant results, pairwise comparisons using the post hoc Dunn test were performed, and p-values were corrected using the Bonferroni–Holm method.

Gravel cores were checked for differences in fines (<2 mm), silt (<0.063 mm) and survival percentages between the L and L + D setups using the independent Two-Sample T-Test. Differences between the L and L + D setups with respect to the two stages, “eyed egg” and “alevin”, were checked for the DO demand, NH₄⁺ and NO₃⁻ fluxes using the Wilcoxon Signed-Rank Test as a non-parametric alternative to cope with the heteroscedasticity and non-normality of the data. For the same reasons, the two stages, “eyed egg” and “alevin”, were tested for any difference in DO demand and NO₃⁻ fluxes using the Wilcoxon Signed-Rank Test; while NH₄⁺ fluxes were tested with the independent Two-Sample T-Test. Finally, the Wilcoxon Signed-Rank Test was used also to check differences in the N-NH₄⁺ and N-NO₃⁻ concentrations between all the inflow and outflow sections grouped together from the mesocosms. All the statistical tests were performed at an alpha level of 0.05. R software [22] was used to perform all the statistical analyses and plot charts using the rstatix [23] and ggplot2 [24] packages, respectively.

3. Results and Discussion

3.1. Egg Respiration and Excretion Rates Measured in Microcosms

The Kruskal–Wallis test indicated that the fish stage significantly affected the DO demand (H(2) = 31, p < 0.001) and NH₄⁺ excretion (H(2) = 30, p < 0.001) (Figure 3). Pairwise comparisons using Dunn’s test showed that the DO demand (mg O₂ egg h⁻¹) in the eyed eggs (294–310 dd) was 0.002 ± 0.0004 mg O₂ egg⁻¹ h⁻¹, which is significantly lower (p = 0.001) than that of the nearly hatched alevins (350–358 dd), respiring 0.016 ± 0.003 mg O₂ egg⁻¹ h⁻¹. In turn, the stage with completely absorbed yolk sacks (454–462 dd) had a DO demand of 0.07 ± 0.007 mg O₂ egg⁻¹h⁻¹, which is significantly higher (p = 0.03) than that of the previous stage.

This marked increment in metabolic activity from the embryonic to larval stage is not surprising since it was observed earlier by Wicket [25] for pink (O. gorbuscha) and coho (O. kisutch) salmons reared at 8 and 5 °C, respectively. Wicket [25] reported metabolic rates for eyed eggs ranging from 0.0006 to 0.0002 mg O₂ egg⁻¹ h⁻¹, sharply increasing by one order of magnitude (0.009–0.01 mg O₂ egg⁻¹ h⁻¹) for newly hatched alevins. Further evidence comes from Alderdice [19], who found that freshly fertilised chum (O. keta) salmon eggs at 10 °C were respiring 0.00093 mg O₂ egg⁻¹ h⁻¹, while hatching larva respiration peaked at 0.0052 mg O₂ egg⁻¹ h⁻¹. Rombough [26] found that pooled rainbow trout embryo and alevin metabolic rates ranged from 0.028 to 0.078 mg O₂ egg⁻¹ h⁻¹ when reared at 6 and 15 °C, respectively. Also, triploid stages of the same species presented a similar trend, spanning from 0.001 (eyed eggs), to 0.004 (hatched alevins), and to 0.03 mg O₂ egg⁻¹h⁻¹ (yolk sack absorbed) [27]. Finally, for Atlantic salmon, the DO demand ranged from 0.0067 [28] to 0.0048 mg O₂ egg⁻¹ h⁻¹ [29] for the hatching larvae under temperatures of 17 and 10 °C, respectively. The variability in the DO flux is thus related to different ontogenetic phases, reflecting different mass and metabolic activities, which, if not clearly pointed out, prevent the making of meaningful comparisons among different works [26,30]. Metabolic rates should be compared when eggs belong to the same stage and when they are exposed to the same level of stress, which are not guaranteed given the different scopes of studies. Indeed, it is well known that activity and stress, though not easily quantifiable or controllable, can greatly affect baseline metabolic rates, attained under ideal conditions [31,32]. For this reason, we believe that the higher variability in the alevin DO respiration rates, especially those of the last batch, can be attributed to the larger size and the increased mobility of the larvae in the restrained closed respirometry chambers with respect to the embryos. Similar problems arise when burrowing macrofauna or when larvae are incubated in the absence of sediments, where they feel uncomfortable and try to dig across the glass walls of the incubation chambers leading to unrealistic, overestimated metabolic activity, largely exceeding the baseline respiration measured when they are within sediments [33]. Another source of variability can be attributed to the usage in some works [19,26] of a mass unit instead of an egg unit, leading to an underestimation of metabolic rates if the inactive mass of the yolk sack is considered in the calculations. This is especially true for the early stages, when the inactive mass of the yolk sack can represent a major fraction of the embryo mass. However, for those works that report metabolic activity in relation to the whole embryo, inter-genera differences in egg size [6,34,35] can explain the variability among respiration rates. Finally, different incubation temperatures inevitably affect metabolic rates and thus the DO demands; for example, Atlantic salmon eggs at the hatching stage incubated at 17 and 5 °C nearly halved their metabolic activity, shifting from 0.0067 to 0.0039 mg O₂ egg⁻¹ h⁻¹ [28].

Ammonium excretion at the eyed stage averaged 0.23 ± 0.07 μg N-NH₄⁺ egg h⁻¹, which is substantially lower (p = 0.001) than the 0.69 ± 0.20 μg N-NH₄⁺ egg⁻¹ h⁻¹ of the alevins with yolk sack, which, in turn, differed significantly from the excretion of the stage with a completely absorbed yolk sack (3.60 ± 2.0 μg N-NH₄⁺ egg⁻¹ h⁻¹, p = 0.04). The increments in metabolic rates, following the ongoing developmental process and the increase in the biomass, explains the increased excretion in the late stages [36]. We speculate that the variability among rates can be justified by the same reasons discussed earlier for DO fluxes, related to the experimental conditions, the size and some stress increasing along with the different stages. Smith [30] was a pioneer in evaluating NH₄⁺ excretion rates from rainbow trout eggs, ranging from 0.1 for early stages to 0.7 μg N-NH₄⁺ egg⁻¹ h⁻¹ for alevins. Such a range of values includes the excretion rate reported for the pre-hatching stages of the same species by Noronha et al. [37] (0.16 μg N-NH₄⁺ egg⁻¹ h⁻¹). Teles [27], incubating diploid and triploid rainbow trout eggs, measured an increment in the NH₄⁺ excretion rates from 0.08 (early eyed) to 0.25 (hatching phase), to a peak of 1.5 μg N-NH₄⁺ egg⁻¹ h⁻¹ (eggs with an exhausted yolk sack). Besides the aforesaid factors dictating diversity in DO respiration activity, for NH₄⁺, an additional variable can be represented through pH changes. Indeed, the NH₄⁺ excretion is primarily driven by the diffusion of the unionised ammonia form (NH₃), owing to a partial pressure gradient, while the ionised ammonium form (NH₄⁺) cannot diffuse across the almost impermeable chorion [37]. Due to its pK of 9.5, the NH₃ will more easily diffuse into acidic compartments [38], and this can double the excretion rates from eggs incubated at pH 10 when they are incubated at lower pH values (e.g., 6 to 8) [37].

3.2. Mesocosms

3.2.1. Setups

With respect to the comparison of the L and L + D treatments, the simulated egg pocket respiration and net ammonium and nitrate fluxes during both the egg and the alevin stages were not significantly different (for DO fluxes: Z_egg = 192, p = 0.36; Z_alevin = 212, p = 0.83; for N-NH₄⁺ fluxes: Z_egg =75, p = 0.86; Z_alevin = 92, p = 0.25; for N-NO₃⁻ fluxes: Z_egg =75, p = 0.89; Z_alevin = 90, p = 0.32). This result can be justified by the “net” nature of the measured fluxes in the gravel cores, integrating the metabolic activity of live eggs, the heterotrophic activity of microbes growing within the substratum matrix or decomposing dead eggs, and the different levels of mortality in the different mesocosms. Similar respiration and ammonium fluxes in the two treatments (L and L + D) may therefore result from the higher metabolic activity of the eggs in treatment L and the higher microbial contribution to the total mesocosm metabolism in treatment L + D.

Nevertheless, during both stages, the DO demand and N-NH₄⁺ fluxes tended to be higher in treatment L (DO demand_egg = 6.7 ± 4.6 mg O₂ mecocosms⁻¹ h⁻¹; N-NH₄⁺ fluxes_egg = 0.12 ± 0.13 mg N-NH₄⁺ mecocosms⁻¹ h⁻¹; DO demand_alevin = 11.1 ± 2.4 mg O₂ mecocosms⁻¹ h⁻¹; N-NH₄⁺ fluxes_alevin = −0.16 ± 0.07 mg N-NH₄⁺ mecocosms⁻¹ h⁻¹) compared to those of treatment L + D (DO demand_egg = 5.3 ± 4.2 mg O₂ mecocosms⁻¹ h⁻¹; N-NH₄⁺ fluxes_egg = 0.08 ± 0.11 mg N-NH₄⁺ mecocosms⁻¹ h⁻¹; DO demand_alevin = 11 ± 2.2 mg O₂ mecocosms⁻¹ h⁻¹; N-NH₄⁺ fluxes_alevin = −0.25 ± 0.2 mg N-NH₄⁺ mecocosms⁻¹ h⁻¹). We speculate that the higher number of live eggs and associated metabolic rates in treatment L together with the undeveloped biofilm at the beginning of the incubation process were the main causes of such differences.

Nitrate fluxes showed a rather erratic trend with a tendency toward higher values during the egg stage for the L + D treatment (N-NO₃⁻ fluxes_L = −0.6 ± 3.0 mg N-NO₃⁻ mecocosms⁻¹ h⁻¹; N-NO₃⁻ fluxes_L+D = −0.1 ± 5.0 mg N-NO₃⁻ mecocosms⁻¹ h⁻¹;). However, this trend was reversed during the alevin phase, when the N-NO₃⁻ fluxes tended to be higher for the L treatment (N-NO₃⁻ fluxes_L = −0.1 ± 6.3 mg N-NO₃⁻ mecocosms⁻¹ h⁻¹; N-NO₃⁻ fluxes_L+D = −3.0 ± 3.4 mg N-NO₃⁻ mecocosms⁻¹ h⁻¹).

Nitrate fluxes were mostly negative, suggesting the consumption of this oxidised ion, even if they were characterised by large variability. We speculate that hypoxic pockets forming around dead eggs collated with biofilm mass may act as local sinks for electron acceptors, depleting the DO and promoting nitrogen loss via denitrification [13,39].

Reconstructed trout egg pockets generally acted as a moderate N-NH₄⁺ source, although not being statistically significant (p > 0.05). Indeed, data pooled from the whole incubation period presented concentrations ranging from 0.290 ± 0.015 to 0.310 ± 0.003 mg L⁻¹ in the input and output sections, respectively. On the other hand, they tended to act as a sink of N-NO₃⁻, with concentrations at the inflow (0.16 ± 0.02 mg L⁻¹) decreasing to 0.130 ± 0.002 mg L⁻¹ at the outflow, which is significant (p = 0.03). Though evidence [39,40,41] points to N-NO₃⁻ being a harmful molecule for the early ontogenetic stages of salmonids, the present levels are far below the thresholds of 20 and 34 mgL⁻¹ proved to be detrimental for species belonging to the same genus of the one used here, such as cutthroat trout (Oncorhynchus clarkii) and chinook salmon (Oncorhynchus tshawytscha) fry [41].

3.2.2. Survival and Substratum Composition

Oxygen concentrations measured at the gravel core outflow averaged 9.7 ± 0.3 mg L⁻¹, a value which is considered highly protective for the intragravel stage under natural conditions [4,42]. However, at the beginning of the experiment, we recorded, with a transient, lower concentrations (6.6 mg L⁻¹), coinciding with the clogging of some tubes of the peristaltic pump due to the accumulation of fine material exported from the cores. Such tubes were promptly cleaned, ensuring homogeneous water flow, and similar episodes did not occur during the rest of the experiment.

We cannot exclude that this initial shortcoming, resulting in a transient decrease in water velocity within simulated egg pockets, can partially explain the variable survival rates of the eggs (6% to 64%) in comparison with the control trays (99 ± 1%). Indeed, it was previously noticed that the swim-up from natural redds, following a successful incubation, occurred at DO values around 7 [8,43], even for those having a sediment composition similar to the one used here [21]. Additionally, in these same environments, flow velocities lower than 500–600 cm/hr severely compromised the survival chances [21]. Although in the present experiment the period of reduced velocity and DO levels lasted only few hours, multiple evidences [3,4,42] suggest that even transient periods of hypoxia can weaken the embryo fitness, concurring with post-hatch mortality.

Another explanation for the poor survival can be found in the fine (<2 mm) content that ranged from 14% to 29% and silt (<63 μm) content that varied from 0.16% to 0.38%. Indeed, the salmonid literature already considers 15–18% in fine content to be unsuitable for successful egg survival [1,5,13,44].

Overall, there was no significant difference between the two setups in terms of fines (t(4) = −0.76, p = 0.49) and silt (t(4) = −0.28, p = 0.79), despite the L treatment showing a lower amount of such fine fractions (<2 mm = 20 ± 6.3; < 63 μm = 0.2 ± 0.07) with respect to the L + D treatment (<2 mm = 23 ± 5 < 63 μm = 0.24 ± 0.13). This slight discrepancy can partially support why the L treatment presented significantly higher (t(4) = 3.64, p = 0.02) survival percentages than the L + D treatment (survival_live: 51% ± 11%; survival_live+dead: 11% ± 6%), assuming that even variations in a few percent of these fractions can dramatically decrease the incubation success [9,45].

Additionally, it must be reminded that the L + D treatment was constituted by a half of dead eggs; indeed, decaying embryos constitute an additional sink of DO, acting also as a source of toxic nitrogen compounds [46,47] such as NH₃. Moreover, dead eggs can represent a fuel for algae and bacterial mat growth that can synergistically act with the fines, cleaving the intragravel matrix and thus preventing proper respiration or upward movement of the embryos and alevins [13]. Our speculations are supported by the fact that during the opening procedure, we did not record any alevin on the top layer of the cores, suggesting that clogged interstices prevented the swim up [8,44]. Such hypoxic bags lead to a cascade effect since larvae excrete, but also accumulate in these clogged spaces, NH₄⁺ and other waste substances, provoking detrimental conditions and egg death in the long run [46,48,49,50]. Indeed, it is widely recognised that biological membranes, like the egg chorion, are highly permeable to the unionised form (NH₃), which constitutes the real threat of the ammonia + ammonium pool (NH₃ + NH₄⁺) and increases with more basic pH levels [36,37,51]. However, according to the pH value measured in the aquarium (nearly 7.4 units), the quantity of N-NH₃ was 1.2 μg L⁻¹, far below the toxicity level (96 h LC50) of 17 μg L⁻¹ quantified for salmonids [47,51]. Nonetheless, we believe that fine particle conglomerations aided by the biofilm presence, lead to an uneven waterflow and O₂ distribution as well as a lowered metabolic waste removal [39,50]. This is in spite of the O₂ and ammonium levels at the outflowing sections being inside the safety ranges. Although this hypothesis remains unproven, it would necessitate a high spatial scale reconstruction of the O₂ distribution and flow map inside the simulated egg pockets [17].

3.2.3. Stages

Upon the stage comparison, the DO demand changed significantly across egg stages (Z = 276, p < 0.001), increasing from 6.0 ± 4.4 mg O₂ mesocosm⁻¹ h⁻¹ for the eyed egg to 11.0 ± 2.3 mg O₂ mesocosm⁻¹ h⁻¹ for the alevin stage (Figure 4). Such an increment is partially explained by the augmented metabolic rates during the embryo development (see the comments on the results of the microcosm experiment) and by the metabolic activity of growing microbial biomass within the substratum. Indeed, the DO demand from the bare gravel re-incubated without eggs was 8.6 ± 1.3 mg O₂ mesocosm⁻¹ h⁻¹ and, although not directly comparable with the entire series, it accounted for 78% of the total mesocosm respiration during the last period of incubation.

Ammonium fluxes changed markedly along the course of the experiment (t(45) = 8, p < 0.001), decreasing form 0.10 ± 0.12 mg N-NH₄⁺ mesocosm⁻¹ h⁻¹ measured at the eyed egg stage to −0.21 ± 0.14 mg N-NH₄⁺ mesocosm⁻¹ h⁻¹ measured at the alevin stage. Although the excretion of nitrogenous wastes was expected to increase along with the embryo development, we speculate that other processes consuming the produced ammonium can contrast its net release outside the system. Such processes can include microbial uptake or nitrification. As the N-NH₄⁺ excretion averaged −0.090 ± 0.014 mg N-NH₄⁺ mesocosm⁻¹ h⁻¹ when only bare gravel was incubated, we speculate that the presence of dead or live eggs together with their biomass supporting heterotrophic activity or excretion supporting nitrification contribute to large microbial growth in the egg pocket proximity [13,39].

Nitrate followed a similar and significant trend (Z = 389, p < 0.04) with higher fluxes measured for the eyed egg stage of −0.4 ± 3.9 mg N-NO₃⁻ mesocosm⁻¹ h⁻¹ as compared to the −1.5 ± 5.2 mg N-NO₃⁻ mesocosm⁻¹ h⁻¹ measured at the alevin stage. This suggests that the incorporation of nitrate into the microbial biomass or denitrification processes were higher during the last phase of the incubation, for which we hypothesised maximum biofilm growth and the presence of hypoxic or anoxic microzones within the mesocosms. In natural environments, anaerobic–aerobic interfaces in the hyporheic zone act as a powerful hotspot of oxidative and reductive processes [52,53]. Thus, the ammonium pool that is successfully oxidised to nitrate in the oxic matrix can fuel the denitrification processes into such hypoxic niches. Net nitrate fluxes in the control cores decreased to −1.15 ± 0.85 mg N-NO₃⁻ mesocosm⁻¹ h⁻¹, compared to the last period. The lower biofilm performances can be attributed to the substratum mixing, oxygenation of the hypoxic niches and decrease in nitrate removing processes.

4. Conclusions

The present study is a pilot investigation of the simulated salmonid egg pocket functioning in terms of the O₂ demand and ammonium and nitrate net fluxes. To our knowledge, this is the first attempt in which fluxes from a reconstructed salmonid egg pockets are measured in an intact substrate setting. Biogeochemical processes are traditionally studied with mesocosm systems in a static water environment, presenting a clear air–water–sediment interface [33,54,55]. The innovative aspect of this study was to adapt such tools to characterise the free-flowing conditions occurring in a lithophilic fish nest, namely a parcel of the hyporheic zone. The aim was reached using whole gravel-packed cores equipped with a flow-through system. It discharged water at a higher rate than usually set in these investigations [55,56,57], in order to meet the O₂ demand of salmonid eggs. The main challenges encountered were related to setting a proper waterflow in order to maintain safe O₂ concentrations in the system, but low enough to have a difference in the inflow and outflow concentrations used to quantify O₂ fluxes. Flow-through systems employing cores of similar volumes are traditionally devised to deliver little amounts of water discharge into the single core [56], and, to accomplish this, only one or two tubes are sufficient. In our case, to provide the required water velocity values, we needed to install five inflow tubes into each core, in order to have a homogeneous distribution of the inflowing water along the whole mesocosms cross-section.

Microcosms were employed to measure net fluxes from single eggs and compare them with the mesocosm setup.

In terms of the O₂ demand, the increment in the cores (mesocosms) follow a parallel trend as the one in the chambers (microcosms); this because of the metabolic intensity increment with advancing developmental stages. Additionally, biofilm growth increases the whole respiration in the cores, becoming predominant in the last phase of the incubation, as remarked by the bare cores (control).

Overall, reconstructed nests acted as an ammonium source, reflecting the increment in the metabolic excretion of larvae at the last stages. Nevertheless, as the incubation progresses, the cores become ammonium sinks, showing a contrasting trend in respect with the eggs alone. This is because of the biofilm formation, which involves an uptake of nutrients for its growth. The same explanation can also be used to justify the increment in nitrate removal, if we assume the formation of hypoxic spots that led to denitrification. Finally, beside flux quantification, the present experimental setup is suitable for a range of other hypotheses testing aspects more directly related to embryo survival. Indeed, with such a configuration, the user has direct control over the environmental variables responsible for survival and development in the gravel matrix. Therefore, factors such as water discharge, temperature, substratum composition and O₂ supply can be adjusted in order to reproduce more or less compromised situations in nature, thus verifying the intragravel quality of spawning beds, as well as measure biogeochemical processes inside gravel egg pockets at a fine scale for not only main chemical compounds, but also other potentially relevant but still not measured elements in the field.