Water quality of spawning grounds constrains
the population dynamics of an emblematic diadromous
species (*Alosa alosa*)

Sarah Bancel D • Jérôme Cachot • Marion Blaya • William Bouyssonnie • Alexandra Coynel • Nicolas Mazzella • Debora Millan-Navarro • Maud Pierre • Olivier Geffard • Éric Rochard

Received: 7 January 2025 / Accepted: 24 March 2025 / Published online: 12 April 2025
© The Author(s) 2025

**Abstract** In this study, the role of water quality has been studied to assess its suitability for the allis shad (Alosa alosa), a diadromous fish for which abundance has drastically declined at the beginning of 2000 and is remaining critically low in the Garonne basin (France). The water quality regarding contamination at spawning grounds and its toxicity for embryos and larvae is one possible explanatory factor for this decline, which has not been investigated. Early life stages of fish are particularly sensitive to

environmental conditions, including water contami-
nation, making them useful for environmental moni-
toring. The ex situ biomonitoring method allows con-
tinuous exposure of organisms to river water while
controlling for confounding factors such as tempera-
ture, oxygen, and photoperiod. Three ex situ expo-
sures of allis shad embryos to the Garonne water were
conducted during its spawning season, in spring 2022
and 2023, under optimal temperature and oxygen
saturation rate. Survival in the control groups dem-
onstrates the value of the ex situ approach, allowing
for the exposure of early life stages in their natural
environment. Results indicated a high mortality rate
in embryos exposed to the Garonne water compared
to control, with mortality occurring during organo-
genesis, which is a particularly critical period of
development. These findings raise concerns about the
suitability of the Garonne water for the early develop-
ment of allis shad and consequently for the popula-
tion dynamics of this species in the Garonne basin.
Moreover, these results confirm previous approaches
of risk assessment and experimentations, suggesting
that water quality and contamination could have an
impact on other fish species.

**Keywords** Allis shad · Garonne river · Ex situ · Embryos · Contamination · Fish population

**Supplementary information** The online version
contains supplementary material available at https://doi.
org/10.1007/s10641-025-01687-7.

S. Bancel (✉) · M. Blaya · N. Mazzella ·
D. Millan-Navarro · M. Pierre · É. Rochard
INRAE Nouvelle-Aquitaine Bordeaux Centre, UR
EABX, 50 Avenue de Verdun, 33612 Nouvelle-Aquitaine,
Cestas Cedex, France
e-mail: sarah.bancel@inrae.fr

J. Cachot
UMR 5805, Université de Bordeaux, EPOC, Univ.
Bordeaux, CNRS, INP Bordeaux, Allée Geoffroy Saint-
Hilaire, 33615 Pessac Cedex, Nouvelle-Aquitaine, France

W. Bouyssonnie · A. Coynel
MIGADO, 18 Ter Rue de La Garonne, 47520 Le Passage,
France

O. Geffard
INRAE Lyon-Grenoble Auvergne-Rhône-Alpes Centre,
UR RiverLy, Auvergne-Rhône-Alpes, 5 Rue de La Doua,
69100 Villeurbanne Cedex, France
## Introduction

Anthropogenic activities are being proven to significantly reduce biodiversity and contribute to the sixth global extinction of species (Ceballos and Ehrlich 2023). In addition to the loss of habitats, overexploitation, invasive species and climate change, it has been shown that chemical pollution could play an important role in biodiversity loss as well (Sigmund et al. 2023). Aquatic ecosystems are particularly impacted by chemical pollution as they are the sink of all human activities (Häder et al. 2020). Thus, aquatic organisms are particularly exposed to chemical pollution; however, links between biodiversity loss or toxic effects and chemical pollution have been under-assessed and require more specific research (Sylvester et al. 2023). Diadromous fish populations are especially vulnerable to anthropic pressures, in particular, because of their specific life cycle shared between marine and freshwater environments. In Western Europe, all 24 diadromous species suffered population losses (Barber-O'Malley et al. 2022): relative abundances for some species have decreased up to 98% on some time series, during the last decades. Several explications for this decline are investigated, such as global changes, overfishing, invasive species, habitat losses, and contamination as well (Jonsson 1999; Limburg and Waldman 2009). Among these factors, the impact of chemical contamination on fish population declines remains poorly understood (Sigmund et al. 2023).

The Garonne basin (south-west France) is no exception, despite hosting still the highest number of diadromous fish species in Europe (Lobry et al. 2003). In this basin, populations of smelt [Osmerus eperlanus (Linnaeus, 1758)], European eel [Anguilla anguilla (Linnaeus, 1758)], European sturgeon (Acipenser sturio Linnaeus, 1758), Atlantic salmon (Salmo salar Linnaeus, 1758), and allis shad [Alosa alosa (Linnaeus, 1758)] are faced with important declines of their abundance those last decades (Pronier and Rochard 1998; Castelnaud 2011; Rougier et al. 2012; Nicola et al. 2018; Dekker 2019). For some of these species, such as allis shad (Alosa alosa), the reasons for the decline remain unclear. Moreover, within the basin, the presence of several metals and persistent organic pollutants was demonstrated in tissues of juveniles and adults of European eel, European sturgeon, twaite shad [Alosa fallax (Lacepède, 1803)],

and thin-lip mullet [Chelon ramada (Risso, 1827)] (Acolas et al. 2020; Daverat et al. 2012; Durrieu et al. 2005; Patey et al. 2017). This shows diadromous fish species are exposed to contaminants of several sources during their life cycle; however, the impact of chemical contamination on those populations is actually not well assessed.

Allis shad (Alosa alosa) is an anadromous fish species (Clupeidae). During spring, sexually mature adults migrate into freshwater to spawn, in the middle watercourse of rivers (Boisneau 1990; Belaud et al. 2001; Acolas et al. 2004). This is where embryos will develop and hatch (Blaya et al. 2022). Juveniles grow in freshwater during the summer (Mota et al. 2015; Baumann et al. 2023) until they initiate a downstream migration in late summer and autumn in estuaries (Lochet et al. 2009) before heading to the sea to achieve their growth (Taverny and Elie 2001; Trancart et al. 2014) until the next cycle. This species was found throughout Europe on the North-East Atlantic coast, from Morocco to Norway. Across its geographical distribution, during spawning migrations, the species was highly exploited, representing a significant economic and social interest (Castelnaud et al. 2001; Rougier et al. 2012). Historically, the most important population of allis shad was located in southwest France, in the Garonne basin where it was considered as a reference population (Baglinière et al. 2003). At the beginning of the 2000s, the population exhibited an important decrease in the number of spawners, leading to the implementation of a fisheries moratorium in 2008 (Rougier et al. 2012). Thus, the population's number of spawners remains quite low. To determine the causes of this decline, several hypotheses were investigated such as global changes (Lassalle et al. 2009; Paumier et al. 2019; Poulet et al. 2021) and overfishing (Rougier et al. 2012) during the adult phase. Regarding the juvenile period, habitat suitability was assessed by several studies (Baumann et al. 2021, 2023) as well as downstream migration (Boussinet et al. 2024). Early life stages (embryos and yolk-sac larvae) are extremely sensitive to environmental parameters and this period is considered as a critical period for populations' recruitment (Kamler 1992). Investigations have been conducted to better study the embryo's development as well as the species' tolerance to temperature in the context of global changes (Jatteau et al. 2017; Blaya
et al. 2022). There is still little explanation for the population's decline and water contamination is one hypothesis to be explored.

Early life stages of fish are also known to be sensitive to pollution and thus are particularly valuable for water quality assessment studies (Von Westernhagen 1988; Bancel et al. 2024). In the Garonne basin, we conducted a study to evaluate the potential toxic risk of pollutants for fish species, especially on allis shad spawning grounds, based on data acquired as part of water bodies monitoring programs (WFD) (Bellier et al. 2024). The results showed that there is a toxic risk for the early life stages of fish in locations close to allis shad spawning areas of the basin. However, this risk was calculated for a limited number of pollutants compared to the wide variety of chemicals putatively present in the Garonne River actually on the market and we used the highest concentrations observed (the worst case). To better integrate the wide variety of contaminants in aquatic ecosystems, as well as their real impact on organisms, methods such as active biomonitoring (ABM) can be used. ABM is defined as the transplantation of calibrated organisms to a potentially contaminated site to observe the biological, physiological, behavioral, and biochemical responses of those organisms to the water quality (Wepener 2013; Bancel et al. 2024).

In this study, an ABM approach was implemented to investigate the responses of allis shad early life stages to the water quality in the spawning area, to assess the suitability of the Garonne's water for the species' early development and the potential role of chemical contamination in the observed lack of recruitment in the catchment. We used a "lab-on-field" approach that allows the exposure of early life stages under controlled conditions of temperature and oxygen saturation rate. Experiments were conducted on the Garonne river, during the allis shad reproduction season in springs 2022 and 2023. Allis shad embryos, from spawners caught during migration on the Garonne River, were directly exposed to the river water throughout their development up to 2 days after hatching, while temperature, oxygen saturation rate, and photoperiod were maintained to optimal values for their development. The control of these environmental parameters and the comparison with controlled exposure allow us to avoid the potential influence of these confounding factors on the interpretation of the results.

## Material and methods

### Description of the study site and experimental setup

From its source in the Spanish Pyrenees to the Gironde estuary on the Atlantic coast, the Garonne River runs 580 km through hills and plains. The Garonne basin has two major urban areas, Bordeaux and Toulouse, and covers around 117,500 km² (Bernard et al. 2019). Its main tributary, the Dordogne River originates in the Massif Central (south-central France) and flows 483 km along the Atlantic coast to the Gironde Estuary, where it merges with the Garonne River. Upstream Toulouse, the hydrology of the Garonne presents a snow regime leading to high flow in spring. Downstream Toulouse, the regime progressively changes in a rainfall regime, whereas in the lower reaches, western winds from the Atlantic Ocean cause high precipitation and cool temperatures.

Approximately 60% of the land is occupied by intensive agriculture. Of this, 50% is under cereal production (60% maize, 30% wheat) and 10% oilseed (sunflower, colza, and soya bean). The remaining agricultural area is dominated by vineyards and fruit trees (Mouton 1990).

Spawning grounds of allis shad (*Alosa alosa*) have been located on both rivers (Fig. 1). Between Malause and Lamagistère, a part of the Garonne flow is diverted on about 15 km to supply the Golfech's nuclear and hydroelectric power plants. Our study site is especially located on this deviated section, immediately upstream of the allis shad spawning area in the Garonne River (Cassou-Leins and Cassou-Leins 1981) (Fig. 1).

We set up a "field lab" at the hydroelectric power plant (44°06'35.6"N 0°51'15.6"E) of Golfech, just above the dam on the deviated river. The most important flow of water runs on this deviated canal (maximum flow rate 540 m³.s⁻¹) compared to 20 m³.s⁻¹ for the historic section. As the electricity is generated on a run-of-river basis, we postulate that the water quality in this deviated channel is similar to the one of the historical channel A filter and decantation tank received water that was directly and continuously pumped from the river at a flow rate of 250 to 300 L per hour. It was made out of a 300-L tank where water was continuously flowing and filtered using blocks of filtration foam with holes ranging from 2 to 4 mm to prevent the introduction of big particles (Fig. 2).
<figure><img src="image_1.png" /><figcaption>**Fig. 1** Location of the studied site in the Garonne basin (from Bancel et al. 2025). Legend for cities on the map of France: B: Bordeaux and T: Toulouse. There are two dams (Golfech and</figcaption></figure>

Malause) one on each section, and six weirs over the historic section (Garonne River)

Every 2 days, these filtration blocks were rinsed with clean water. Subsequently, water flowed by gravity in the “field-lab” into an 80-L initial distribution tank for embryo exposure. Water temperature was regulated using temperature controllers (TECO® TK). Water was then pumped into six exposure incubators, each with a capacity of 1 L, modified from (Delage et al. 2014). To provide bottom-up water circulation, the incubators are made of polyethylene terephthalate glycol (PETg) modified with an additional stainless-steel grid on which the embryos were placed. Within the field-lab, control exposure was carried out in parallel using a closed exposure circuit receiving dechlorinated tap water (tap water oxygenated for 24 h prior to exposures) (Fig. 2).

The exposure system was rinsed before each experiment. In both conditions, the oxygen saturation rate was kept above 90% and the temperature was adjusted to 20 ±1 °C, which is the optimum for allis shad embryonic

development (Jatteau et al. 2017). The photoperiod in the field lab was light:dark 14 h:10 h, as it was outdoors at the time. Before being returned to the river, the water leaving the exposure systems was collected in an exit tank, which was fitted with a filtration block to prevent organism escapes. With the exception of the filtration tank (a plastic rainwater collection tank), where water from the Garonne has been cycled for a full week before introduction, all of the pipelines, faucets, and tanks used are approved for use with food, to prevent the release of potentially toxic compounds through the equipment used.

## Test with allis shad embryos

### Water parameters

For each experiment, five incubators were dedicated to embryo exposure, and one incubator was used to
<figure><img src="image_2.png" /><figcaption>**Fig. 2** Diagram of general hydraulic circulation within the field-lab for the exposure of allis shad embryos to the Garonne water and control circuit. The distance between the Garonne and the field-lab was around 20 m. Adapted from Bancel et al. (2025)</figcaption></figure>

monitor water parameters in each condition (temperature was measured using Tinytag Aquatic 2 data loggers, and conductivity, pH, and oxygen saturation rate were measured using multiparameter probe WTW MultiLine® Multi 3630 IDS). Every hour, the temperature was recorded, and every day at the same time in the morning, the pH, conductivity, and oxygen saturation rate were measured. JBL Pro Aquatest Kits® were used for phosphates (PO₄³⁻) and nitrogen forms (NH₄⁺, NO₃⁻, and NO₂⁻). Every 2 days, NH₄⁺, NO₃⁻, and PO₄³⁻ were measured in addition to the daily monitoring of NO₂⁻. Every day, turbidity was measured using a portable turbidimeter (Hach® 2100).

### Embryo-toxicity test

Experiments were conducted in 2022 and 2023. Allis shad embryos resulted from the assisted reproduction of group spawners captured during their spawning

migration in Garonne at Golfech fish lift and provided by the NGO MIGADO. Fish were captured in a fish trap, transported to a nearby MIGADO facility, and injected with LHRH. Spawners of both sexes are then placed together in circular spawning tanks with a strong current to induce spawn (Jatteau and Rouault 2004). Reproduction occurs during the night following the injection, and embryos are collected the following morning. Embryos were not disinfected before exposure. The age of embryos is estimated in hours post-fertilization (hpf) according to the development table of the species (Blaya et al. 2022). After this, a minimum of 1000 freshly fertilized embryos were carried to the experimental set-up for exposure. One hundred embryos were then randomly placed in each exposure incubator (for controls and Garonne). Mortality, survival, and hatch were monitored twice a day, and dead individuals were immediately removed. When at least a portion of the larval body was
visible outside the chorion, the larvae were considered half-hatched.

Using survival, mortality, and hatch data at each time point, the following results were calculated by dividing each time by the total number of viable embryos at the beginning of the experiment.

- The percentage of embryonic survival was obtained with the number of viable embryos at each time point divided by the number of viable embryos at the beginning of the experiment.

- The percentage of hatches was obtained by dividing the number of hatches at each time point divided by the number of viable embryos at the beginning of the experiment.

- The percentage of half-hatched larvae was calculated with the number of embryos when at least one part of the larval body was outside the chorion at each time point, divided by the number of viable embryos at the beginning of the experiment.

Final survival (total number of larvae), cumulated embryonic mortality, cumulated larval mortality, and remaining viable embryos were calculated as a percentage compared to the number of embryos at the beginning of the experiment.

Exposure lasted until 2 days after the hatch peak was attained (more than half of the embryos hatched). Ten hatched larvae per replicate were used for biometric measurements at the end of the experiment. A Dino Lite® digital microscope was used to acquire pictures at a magnification of 32.5 ×, and the ©ImageJ software was used to measure the yolk sac surface, head length, and overall length.

## Statistics

The R software was used to carry out the statistical analysis (http://cran.r-projet.org/). Statistical significance was set at 5%. For every experiment,

comparisons were made between the control and Garonne conditions. To compare control and Garonne conditions for allis shad embryonic mortality and hatch as a function of time, Kaplan–Meier analysis (log-rank test) was carried out. The analysis included events of hatch or embryonic death (for embryonic survival and hatch dynamic, respectively). To compare conditions for the biometric measures (total length, head length, yolk-sac surface, and head/total length ratio), non-parametric tests (Mann–Whitney) were performed to compare conditions, as there was no homogeneity of variance for all measures between conditions (Levene test). There were no significant differences between replicates in each condition.

## Results

Three distinct experiments were conducted in this study, in 2022 and 2023, and each experiment was realized on embryos of different batches from different spawners’ groups (Table 1).

### Water parameters

Temperature was within the targeted range for the three experiments (20 ± 1 °C) as well as oxygen saturation rate (> 90%) (Table 2). Conductivity, pH, nitrogen forms, and phosphate values are stable over time and did not reach toxic values for allis shad embryos.

### Toxicity for allis shad embryos

#### General survival and hatch rates

In the control condition, for each experiment, survival of allis shad embryos at the end of the experiment

<table><caption>**Table 1** Fertilization date, age of embryos in hours post-fertilization (hpf) at the beginning of exposure, and characteristics of spawners for each experiment conducted</caption><thead><tr><td>Experiment</td><td>Date</td><td>Age of embryos (hpf)</td><td>Total number of spawners</td><td>Number of males</td><td>Number of females</td></tr></thead><tbody><tr><td>1</td><td>May 26, 2022</td><td>16</td><td>21</td><td>12</td><td>9</td></tr><tr><td>2</td><td>June 04, 2022</td><td>16</td><td>10</td><td>6</td><td>4</td></tr><tr><td>3</td><td>May 16, 2023</td><td>8</td><td>33</td><td>18</td><td>15</td></tr></tbody></table>
<table><caption>**Table 2** Physico-chemical parameters measured during allis shad embryo exposure</caption><thead><tr><th rowspan="2">Experiment</th><th rowspan="2"></th><th colspan="2">Experiment 1</th><th colspan="2">Experiment 2</th><th colspan="2">Experiment 3</th></tr><tr><th>Control</th><th>Garonne</th><th>Control</th><th>Garonne</th><th>Control</th><th>Garonne</th></tr></thead><tbody><tr><td>Temperature (°C)</td><td>Mean ± sd</td><td>20.7 ± 0.35</td><td>20.0 ± 0.69</td><td>20.9 ± 0.50</td><td>20.9 ± 0.53</td><td>20.2 ± 0.2</td><td>20.1 ± 0.2</td></tr><tr><td>pH</td><td>Mean ± sd</td><td>8.2 ± 0.2</td><td>7.8 ± 0.1</td><td>8.2 ± 0</td><td>7.9 ± 0.1</td><td>8.2 ± 0.04</td><td>8.1 ± 0.02</td></tr><tr><td>Conductivity (µS/cm)</td><td>Mean ± sd</td><td>261.4 ± 21.5</td><td>234.7 ± 10.3</td><td>251.1 ± 2.4</td><td>233.3 ± 9.3</td><td>305.8 ± 1.9</td><td>267.2 ± 3.8</td></tr><tr><td>Oxygen saturation rate (%)</td><td>Mean ± sd</td><td>96.5 ± 0.8</td><td>87.4 ± 7.1</td><td>98.4 ± 0.5</td><td>90.3 ± 3.1</td><td>99.3 ± 0.3</td><td>101.0 ± 0.5</td></tr><tr><td>NH<sub>4</sub> (mg/L)</td><td>Max value</td><td>&lt; 0.05</td><td>&lt; 0.05</td><td>&lt; 0.05</td><td>&lt; 0.05</td><td>&lt; 0.05</td><td>&lt; 0.05</td></tr><tr><td>NO<sub>3</sub> (mg/L)</td><td>Max value</td><td>1</td><td>1</td><td>1</td><td>1</td><td>&lt; 0.5</td><td>1</td></tr><tr><td>NO<sub>2</sub> (mg/L)</td><td>Max value</td><td>0.1</td><td>0.2</td><td>0.025</td><td>0.025</td><td>&lt; 0.01</td><td>0.1</td></tr><tr><td>PO<sub>4</sub> (mg/L)</td><td>Max value</td><td>&lt; 0.02</td><td>&lt; 0.02</td><td>&lt; 0.02</td><td>&lt; 0.02</td><td>&lt; 0.02</td><td>&lt; 0.02</td></tr></tbody></table>

Values for temperature, pH, conductivity, and oxygen saturation rate are mean ± sd. Values for NH₄, NO₃, NO₂, PO₄, and turbidity are the maximum values obtained

was consistent between replicates (Fig. 3). However, the mean hatching rate was different from one experiment to another with 75.2% for experiment 1, 57.4% for experiment 2 (season 2022), and 93.6% for experiment 3 (season 2023).

In the Garonne condition, high mortality occurred in the three experiments (means of embryonic mortality were 79.4%, 84.6%, and 91.4% for experiments 1, 2, and 3, respectively). As a result, the percentage of hatched larvae was much lower in the Garonne condition than in the control. For each experiment, the mean of hatched larvae in the Garonne condition was

13.4%, 11.4%, and 3.4% for experiments 1, 2, and 3, respectively.

## Embryonic survival

Patterns of embryonic survival dynamics (from the beginning until the end of the experiment) are similar in the three exposures with a survival probability in the Garonne condition which is statistically much lower than in the control, for the three experiments (*p*-value < 0.05, Kaplan–Meier analysis) (Fig. 4). We however observe a lower survival in control of

<figure><img src="image_3.png" /><figcaption>**Fig. 3** Final survival of embryo-larvae of *Alosa alosa* following exposure to control water or Garonne river water. One hundred embryos were placed in each replicate at the start of exposure. Control replicates are C1, C2, C3, C4, and C5. Garonne replicates are Ga1, Ga2, Ga3, Ga4, and Ga5. Gray: cumulated embryonic mortality, pink: larval mortality, blue: remaining viable embryos, green: larval survival at the end of the experiment</figcaption></figure>
experiment 2. Most of the mortality occurred during the embryonic period, particularly between 30 and 60 hpf for the three experiments.

## Dynamic of hatch

The dynamic of hatches during the exposure is similar between the three experiments, with a significantly higher hatch rate in the control condition than in the Garonne condition (p-value < 0.05) (Fig. 5). It should be noted, however, that the start of hatching occurred slightly earlier in the Garonne condition than in the control (12 to 24 h in advance).

## Biometrics

At the end of exposure, the biometric of surviving larvae was measured. We used 10 larvae per replicate for control (in total 50 larvae by experiment), and the remaining larvae for the Garonne condition: 34, 14, and 10 for experiments 1, 2, and 3, respectively (Table 3). No developmental malformations were observed. There were significant differences between conditions following statistical tests for some parameters. The total body length was lower in the Garonne condition than in the control of the first experiment

and this difference was statistically different for experiments 2 and 3. Head length was also smaller in the Garonne than in the control (significant difference for experiments 1 and 3). The ratio of head length to total length was statistically different in experiment 3. Finally, the yolk-sac surface was larger for larvae exposed to the Garonne River than to control water (significant difference for experiments 1 and 3). However, differences between conditions were not particularly important.

## Discussion

### Contribution of the exposure method

Temperature and oxygen saturation rates are known to greatly influence the early development of fish (Kamler 2002). During experiments, both parameters were controlled and maintained at optimal values for allis shad embryo development, allowing the comparison between control and Garonne conditions (Jatteau et al. 2017). Water parameters, such as pH, conductivity, nitrogen forms, and phosphate, did not reach values that could be toxic for allis shad embryos compared to control and according to OECD guidelines

<figure><img src="image_4.png" /><figcaption>**Fig. 4** Embryonic survival probability (in percentage) of *Alosa alosa* following exposure to control water and Garonne river water. One hundred embryos were placed in each replicate at the start of exposure. Vertical bars correspond to events</figcaption></figure>

of the hatch, which were then excluded from the analysis. P-value < 0.05 between both conditions for each experiment (Kaplan–Meier analysis)

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 5** Cumulated hatches as a percentage of the total number of embryos of *Alosa alosa* at the beginning of experiments, following exposure to control water and Garonne river water. One hundred embryos were placed in each replicate at the start</figcaption></figure>

of exposure. Results are expressed as mean ± SD. *P*-value <0.005 for both experiments (Kaplan–Meier analysis comparing conditions for each experiment)

<table><caption>**Table 3** Biometric endpoints in *Alosa alosa* larvae after exposure in the Garonne River</caption><thead><tr><th rowspan="2">Experiment<br>Condition</th><th colspan="2">Experiment 1</th><th colspan="2">Experiment 2</th><th colspan="2">Experiment 3</th></tr><tr><th>Control</th><th>Garonne</th><th>Control</th><th>Garonne</th><th>Control</th><th>Garonne</th></tr></thead><tbody><tr><td>Number of individuals</td><td>50</td><td>34</td><td>50</td><td>14</td><td>50</td><td>10</td></tr><tr><td>Total length (mm)</td><td>9.4 (1.3)</td><td>9.0 (1.1)</td><td>8.9 (0.7)</td><td>8.4 (0.2)</td><td>9.0 (0.4)</td><td>8.6 (0.3)</td></tr><tr><td>Head length (mm)</td><td>1.5 (0.1)</td><td>1.4 (0.1)</td><td>1.4 (0.1)</td><td>1.4 (0.1)</td><td>1.7 (0.1)</td><td>1.5 (0.1)</td></tr><tr><td>Ratio head/total length</td><td>0.16 (0.02)</td><td>0.15 (0.01)</td><td>0.16 (0.01)</td><td>0.17 (0.02)</td><td>0.18 (0.02)</td><td>0.18 (0.01)</td></tr><tr><td>Yolk-sac surface (mm²)</td><td>0.0 (0.0)</td><td>0.4 (0.3)</td><td>0.5 (0.2)</td><td>0.6 (0.3)</td><td>0.3 (0.2)</td><td>0.4 (0.2)</td></tr></tbody></table>

Biometric results are expressed as the median (interquartile range). Values in italics are significantly different from the controls

for toxicity tests using early life stages of fish, even if pH and conductivity values were generally lower in the Garonne condition (OECD 1998). In controls, survival and time for development at 20 °C were consistent with research previously conducted (Jatteau et al. 2017; Blaya et al. 2022). Thus, this field experimental method is valuable for the exposure of sensitive organisms such as fish embryos and enables to investigate their adaptive and deleterious responses to water quality and contamination.

The difference in final survival in controls, especially for the second experiment, shows the variability of egg quality between experiments, which was

previously observed in the literature (Jatteau et al. 2017; Blaya et al. 2022). As spawners’ groups were different between experiments and coming from the wild, the quality of gametes can differ depending on their number and health state. However, the controls used for each experiment ensure that this variability does not introduce uncertainties in the result interpretation.

### Water quality and toxicity for organisms

The three experiments carried out in 2022 (1 and 2) and 2023 (3) highlight a very high mortality rate for
embryos exposed to the Garonne River water, while mortality was much lower in the controls. Mortality occurs mostly between 30 and 60 hpf, which corresponds to the organogenesis period (Blaya et al. 2022). A peak of mortality occurring during this specific phase of development can indicate a specific sensitivity window to water quality and chemical compounds (Von Westernhagen 1988). Additionally, at the end of the experiment (6 to 7 days after fertilization), larvae exposed to the Garonne River tend to be smaller with a bigger yolk sac than the control larvae. This should result from different growth conditions, with slower development in the Garonne than in the control. Reduced length at hatch has already been observed on brown trout (*Salmo trutta* Linnaeus, 1758) embryos exposed to HAPs (Luckenbach et al. 2003) and in Japanese medaka [*Oryzias latipes* (Temminck & Schlegel, 1846)] embryos exposed to naphtalenic acid (Farwell et al. 2006). Metals can also cause reduced length at hatch: it has been shown on herring embryos exposed to metals such as copper, cadmium, and zinc (Von Westernhagen 1988). Reduced consumption of the yolk sac can indicate a metabolic disturbance, caused by chemical compounds. This has been shown in persistent organic pollutants on salmonids (Chapman and Shumway 1978) and benzene and cadmium on Pacific herring embryos (*Clupea pallasii* Valenciennes, 1847) (Struhsaker 1977; Alderdice and Rosenthal 1979). Finally, during our experiments, hatching seemed to occur earlier and at a smaller size in the Garonne River than in the controls, which can indicate stress such as chemical exposure. Early hatching was also observed in embryos exposed to metals (Von Westernhagen 1988) and active endocrine compounds, such as beta-stosterol (Orrego et al. 2011). However, in contrast with the high embryonic mortality observed in the Garonne condition, we did not observe any developmental malformations and biometric results do not indicate important differences between conditions and thus any future viability impairment of allis shad larvae.

The human activities in the Garonne basin are diverse. Due to industrial and mining activities, the presence of metals in the Gironde basin is already well assessed (Grousset et al. 1999; Coynel et al. 2009). Agriculture is highly developed too, and pesticides are regularly found in the Garonne River and can represent a toxic risk for aquatic organisms

(Faggiano et al. 2010; Bernard et al. 2023). Chemical analyses were conducted to monitor 63 pesticides and 15 trace metals in water samples from the 3rd experiment (detailed protocols are available in Bancel et al. 2025). Results obtained did not show the presence of toxicants in concentrations that could explain the high mortality of allis shad embryos observed in the Garonne River in 2023 (Supplementary Information, Tables S1 and S2). These results are similar to those from a different study conducted several weeks before, during spring 2023, showing that among the monitored pollutants, none was present in important concentrations during experiments in the Garonne River (Bancel et al. 2025). It has also been shown that other organic contaminants are present in the basin, such as perfluoroalkyl substances (Munoz et al. 2015, 2019) and drugs, such as hormones and antibiotics (Destrieux et al. 2017). As a result, there are many types of contaminants potentially present, and in mixture, in the Garonne River that can have an effect on allis shad embryos, which could be interesting to monitor in further studies.

Allis shad spawning grounds are close and upstream to those of other species, such as the European sturgeon (*Acipenser sturio*) (Jego et al. 2002). For this species, previous work has shown that contamination could have a significant impact on the early life stages of development of *A. sturio* and Japanese medaka. Particularly, it has been demonstrated that exposure of embryos to sediments of spawning grounds of the Dordogne and the Garonne rivers could cause significant developmental abnormalities and developmental defects (Delage 2015). Additionally, it has been demonstrated that chemical contamination on spawning grounds of the Garonne basin presents a toxic risk for early life stages of allis shad as well as European sturgeon (Bellier et al. 2024). A previous study was conducted in 2023 on the same site using the same experimental set-up used a model fish species, the Japanese medaka. Exposures with this model species with a completely different ecology and origin (small size, exotic species reared in laboratory) showed very similar results, particularly high embryonic mortality occurring during the organogenesis period (Bancel et al. 2025). All these results point out that other fish species in the Garonne, for which no monitoring exists, could also be impacted by chemical contamination.

<figure><img src="image_7.png" /></figure>
Implications for the allis shad population

Previous experiments of Blaya et al. (unpublished
data) using a close exposure method (ex situ approach
without temperature control) showed similar survival
rates of allis shad larvae 2 days after hatch exposed
to the Garonne and Dordogne river (approximately
25% on the Garonne; and between 25 and 50% on
the Dordogne depending on experiments). Addition-
ally, a recent study evaluating the toxic risk of chemi-
cals on allis shad on both spawning grounds showed
that the toxic risk was lower on the Dordogne River
than on the Garonne (Bellier et al. 2024). Within the
Garonne basin, spawning grounds of allis shad are
present on the Garonne and Dordogne rivers. Thus,
it seems especially interesting to do the same experi-
ment on the spawning grounds of the Dordogne River
to assess whether water quality could be more favora-
ble to maintain the population in the Garonne basin.

This high mortality at an embryonic stage raises
concern about the suitability of spawning grounds
for the development and recruitment of the popula-
tion in the Garonne River. Recruitment in popula-
tion dynamics refers to the number of new recruits
that result from the reproduction of a specific num-
ber of spawners (Gaillard et al. 2008). For the allis
shad population in the Garonne basin, it is possible
to assess recruitment by estimating the number of
spawners present on spawning grounds throughout
the reproductive season and the number of juveniles
in the estuary during their downstream migration.
Previous studies suggested an insufficient recruit-
ment of the population, explaining the still low levels
of spawners in the basin despite the fishery morato-
rium (Rougier et al. 2012). Insufficient recruitment
indicates a lower number of juveniles compared to
the number of spawners in reproduction, indicating
a survival issue between these two life stages. The
ecology of allis shad juveniles constitutes a develop-
ment period that is still poorly understood. Addition-
ally, we know that spawners are physiologically able
to produce large numbers of eggs and that these eggs
can develop properly in control conditions (Jatteau
and Rouault 2004). This study constitutes the first
key finding explaining the limited recruitment of the
population, our results suggest poor water quality for
embryonic development and it seems imperative to
find the reason of this significant mortality.

Beyond the Garonne basin, this experimental
approach could be particularly helpful for assessing
the suitability of habitats of the species for conserva-
tion purposes. After having disappeared in the Rhine
River due to overfishing and habitat degradation
(Groot 1990), the allis shad has been reintroduced
since 2008 with larvae reared from the MIGADO
facility. Results are promising as adults in migration
were observed in freshwater (Hundt et al. 2015). The
deployment of the ex situ exposure method in the
Rhine River could help assess the quality of spawn-
ing groundwater and the general suitability of the
Rhine basin for the stocking of allis shad. It has also
been predicted that allis shad could expand its repar-
tition zone northward in response to climate change
(Rougier et al. 2015). In this context, our experimen-
tal approach may allow us to predict and assess the
suitability of future habitats for the species, by assess-
ing the survival of early life stages of allis shad when
exposed directly to the natural environment.

Conclusion

To conclude, this study shows a high impact of the
water quality of the Garonne River on allis shad embryo
development, in three different experiments exposed on
two consecutive years during the reproduction period
of the species. The timing of mortality, as well as bio-
metric measures, suggest the presence of chemical
compounds in the spawning groundwater. Such results
raise questions about the potential role of pollution in
the decline of this species and the actual suitability of
the river for the maintenance of the population within
the basin. It appears crucial to delve into the causes of
this mortality, especially chemical contamination in the
basin and its potential sources. Further investigation
on other spawning grounds of allis shad in the basin is
required, as well as spawning grounds of other diadro-
mous species. Finally, this experimental approach can
be particularly useful for evaluating habitats suitability
for endangered fish species conservation.

**Acknowledgements** We want to thank the Adour-
Garonne Water Agency (AEAG) and two INRAE (Institut
National de Recherche Pour l'Agriculture, l'Alimentation et
l'Environnement) units EABX and Riverly for providing finan-
cial support for this work. We also want to thank the associa-
tion MIGADO and EDF (especially the hydroelectric Power
Plant of Golfech) for providing facilities for these experiments.
This work was also supported by the XPO Scientific Infrastructure of the EABX Research Unit.

We thank Romaric Le Bahr, Baptiste François, and Stéphane Bons for their assistance in these experiments and data analysis. Thanks to Helen Mc-Combie Boudry from the Université de Bretagne Occidentale for editing the English syntax and grammar.

**Author contributions** Conceptualization: SB, JC, MB, AC, NM, OG, and ER.

Methodology: SB, JC, AC, DMN, NM, MB, OG, and ER.

Formal analysis and investigation: SB, AC, DMN, NM, and MP.

Writing—original draft preparation: SB.

Writing—review and editing: JC, MB, WB, AC, DMN, NM, MP, OG, and ER.

Funding acquisition: OG and ER.

Resources: WB and MP.

Supervision: JC, OG, and ER.

**Funding** This work was supported by the Adour-Garonne Water Agency (AEAG) under the grant no. 2021/933 and two INRAE (Institut National de Recherche Pour l'Agriculture, l'Alimentation et l'Environnement) units, EABX and Riverly.

**Data availability** Data used in this study can be made available from the corresponding author on request.

## Declarations

**Ethical approval** We declare that all experiments were conducted on embryos and yolk sac larvae, which are exempt from the EU's 2010/63/EU animal testing regulations.

**Competing interests** The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sarah Bancel reports financial support was provided by Adour-Garonne Water Agency (AEAG).

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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