Testing resistance of Daphnia magna in various environments

doi:10.21203/rs.3.rs-5204857/v1

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Testing resistance of Daphnia magna in various environments

https://doi.org/10.21203/rs.3.rs-5204857/v1

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Environmental pollution is a critical threat to aquatic ecosystems, particularly in regions subjected to industrial and urban pressures. This study examines the application of Daphnia magna as a bio-indicator species for detecting pollution in various Algerian water sources, including desalination water, wastewater, oueds, and industrial effluents. The primary objective is to assess water quality at these key locations by analyzing the effects of different effluents on the life history traits of Daphnia magna. This research is essential for advancing environmental monitoring and pollution assessment, especially as anthropogenic impacts on aquatic ecosystems intensify. Addressing existing knowledge gaps, the study focuses on specific pollutants in Algerian waters and their impact on Daphnia magna. The methodology involves chronic and acute toxicity tests, offering a thorough evaluation of water quality through multiple exposure scenarios. Key findings reveal significant adverse effects of effluents, such as reduced size at first reproduction, decreased longevity, and altered reproductive parameters. These results underscore the pronounced sensitivity of Daphnia magna to pollution, reinforcing its effectiveness in environmental monitoring. The implications suggest that water quality at the studied sites is compromised, with potential consequences for broader aquatic ecosystems, emphasizing the necessity for ongoing, systematic monitoring and intervention.

Daphnia magna

bio-indicator

water quality

pollution detection

aquatic ecosystems

Algeria

In a context where the preservation of the environment and the protection of water resources are major issues, the assessment and detection of pollution in various water samples is of critical importance. Our study focuses on the assessment of water quality in several treatment plants, including desalination plant, wastewater treatment plants, Oued and industrial waters. Our approach is based on the use of Daphnia magna as a bio-indicator in chronic and acute tests. Water, is a limited natural resource on Earth. Faced with this alarming situation, bio-indication offers a promising approach to monitoring and controlling the state of the environment (Rivière, 1998). By using sentinel organisms, such as micro-crustaceans, as biological indicators, it is possible to assess the impact of pollutants on aquatic ecosystems in a comprehensive and integrated way.

Daphnia magna (Straus, 1820), a freshwater microcrustacean of the Cladocera order, is integral to aquatic food chains with a crucial role. It consumes phytoplanktonic algae and serves as prey for fish and aquatic larvae, making it essential in lentic ecosystems. This species is widely used as a reference model in bioassays for assessing the potential toxic effects of chemical substances due to its small size, short life cycle, asexual reproduction, and high fecundity (Florian Parisot, 2015; Orsini et al., 2017). Thriving in lakes and ponds with moderate light, temperatures of 15-25°C, and slightly alkaline to neutral waters (pH 6.5-8.5) with adequate calcium (Hooper, 2008), it feeds on suspended particles, periphyton, and detritus. Despite its vulnerability to fish predation, it is protected from smaller invertebrates and copepods (Brooks & Dodson, 1965; Dodson & Frey, 2001). The species also hosts diverse microbiota that impact its health (Callens et al., 2016). Its body, covered by a chitinous cuticle, features lateral folds and five pairs of thoracic appendages (phyllopods) for filtering food. The biramous antennae facilitate swimming, giving rise to its nickname "water fleas" (Ebert, 2005). The feeding process involves filtering particles with thoracic appendages, transporting them to the mouth for digestion through the esophagus, midgut, and hindgut. It primarily consumes phytoplankton but can also ingest bacteria and fungi, with its color varying based on diet (Ebert, 2005). Reproductive strategies include cyclical parthenogenesis under favorable conditions and sexual reproduction during stress, producing resistant ephippial eggs. Females are generally larger (3-5 mm) than males (2-3 mm), with males having longer antennules for grasping females and specialized thoracic appendages (Ebert, 2005; Lampert, 2011; Zeman, 2008). The life cycle comprises three stages: embryonic (3 days), juvenile (6 days), and adult (until death), with females releasing eggs every 2-3 days under optimal conditions and males appearing during sexual reproduction influenced by environmental stressors (Hebert, 1978; Ebert, 2005; Zeman, 2008).

Effective bio-indicators exhibit moderate tolerance to environmental variability, enabling them to detect changes while providing a reliable measure of biotic responses (Holt et al., 2010). They are chosen for their sensitivity to specific pollutants, serving as proxies for broader ecological impacts (Van der Oost et al., 2003). Bio-indicators integrate biological, physical, and chemical data, revealing changes in individual fitness, population dynamics, and ecosystem processes, and guiding management actions for sustainability (Holt et al., 2010). However, challenges include distinguishing natural variability from anthropogenic effects and the influence of factors like disease, competition, and habitat specificity on indicator species (Holt et al., 2010). Daphnia magna reacts to pollutants through complex mechanisms. Heavy metals such as cadmium, lead, and mercury accumulate in its tissues, leading to bio-magnification and oxidative stress, disrupting growth, reproduction, and metabolism (Schwartz et al., 2013; Wang et al., 2023; Georgieva et al., 2019). Persistent organic pollutants (POPs) like PCBs accumulate in lipids, disrupting metabolic functions and reproduction (Egle Kelpsiene et al., 2023; Vera Kovacevic et al., 2018). Eutrophication and pesticide exposure impact Daphnia through acute toxicity, reduced reproduction, and altered growth (Wang et al., 2023; Chen et al., 2024). Extreme pH levels affect carapace strength and physiological processes (Glover et al., 2005; Weber et al., 2009). pharmaceuticals and personal care products disrupt hormonal regulation, affecting reproduction (Sousa et al., 2021). The behavioral changes in Daphnia due to pollutant exposure include altered swimming patterns, feeding behavior, and escape responses. For instance, sub-lethal concentrations of copper reduce swimming speed and impair phototactic behavior, affecting predator evasion and food finding, leading to reduced fitness and survival (Gerhardt, 1995). Pollutants cause hypoactivity and alter light responses, with effects varying by contaminant concentration (Egan et al., 2023). Xenobiotics like metals, pesticides, nanoparticles, and cyanotoxins can adversely affect swimming behavior, impacting health and population dynamics (Chevalier et al., 2014). Pollutants influence swimming ability, with effects depending on species, dosage, exposure duration, and type of pollutant (Pan et al., 2016). Endocrine-disrupting chemicals, such as bisphenol A (BPA), can alter sex ratios, delay maturation, and reduce fecundity, while heavy metals and pesticides can cause developmental abnormalities and growth defects (Kim et al., 2011; Flaherty et al., 2005). At the molecular level, pollutants induce stress responses in Daphnia, including inhibition of essential proteins and enzymes by heavy metals, and disruption of cellular processes by pesticides and nanoplastics (Xu et al., 2018; Arao et al., 2020). Daphnia serve as effective bio-indicators due to their sensitivity to pollutants and environmental changes, with their reproductive and physiological responses providing early warning signals of water quality deterioration (Rodrigues et al., 2022). Monitoring Daphnia populations alongside water quality parameters like pH, temperature, and contaminants can offer insights into ecosystem health (EPA, 2002). Daphnia play a crucial role in aquatic ecosystems, influencing algae populations and providing food for higher trophic levels. Their decline can lead to algal blooms and disrupt ecosystem balance (Lampert, 2011). Regulatory agencies, including the EPA and the European Commission, use Daphnia toxicity tests in water quality assessments to protect aquatic life and ensure sustainability (EPA, 2002; European Commission, 2003). All of the previous information are what led us to choose daphnia magna for our study.

Our research focuses on the impact of different effluents on Daphnia magna and aims to improve water quality management and monitoring. The study has several objectives: Scientific: Understand the impact of water effluents on Daphnia magna, including the mechanisms of action and sensitivity to various effluents. Methodological: Develop and refine eco-toxicological risk assessment methods, improve test reliability, and harmonize protocols. Management: Establish water quality standards, implement monitoring tools, and propose strategies to minimize environmental imp acts.To achieve our objectives, we adopt a multidisciplinary approach combining biological, eco-toxicological and environmental analysis methods. We are using Daphnia magna, a model organism widely recognized for its sensitivity to environmental contaminants, in chronic laboratory and field tests. This thesis will address the importance of maintaining good ecological status in aquatic ecosystems, in particular by guaranteeing good water quality. We will explore the concept of bio-indicators and highlight the importance of Daphnia magna in assessing water quality. Finally, we will present the objectives and methodological approach of our study, aimed at contributing to a better understanding and management of water pollution.

Geographic distribution

Daphnia magna is widely distributed in Europe, including the Mediterranean and North Africa (Eugeniya et al., 2018). It is found in freshwater systems throughout Europe (Fischer et al., 2011) and in North America, though often due to human introduction. In Africa, it occurs in both North and South Africa. In Algeria, it is present in various freshwater habitats including Lake Taza, Lake Oubeira, and the Sebaou and Soummam rivers (Laboratory of Wetland Conservation). The species is also found in the Middle East, Central Asia, and parts of Siberia, China, and Japan (Eugeniya et al., 2018). with the exact distribution illustrated in the following two maps: one showing the global distribution and the other focusing on the regional distribution.

This study was conducted at the National School of Marine Sciences and Coastal planning ENSSMAL, specifically in the Marine Chemistry Laboratory 1 LBCM1 and the aquaculture farm of the School. The research focused on monitoring the cultivation of the species Daphnia magna and examining its behavior in various water samples under different conditions of stress. The goal was to detect the presence or absence of pollution in the collected water based on the concept of bio-indicator in different samples from various environments: Fouka Tipaza desalination plant, Beni Merad wastewater treatment plant, and Oued Chiffa oued sebaou and tonic industry.

2.1 Study sites

Fouka Seawater Desalination Plant: Located on the border between Fouka and Douaouda municipalities in Tipaza province, this plant covers 8 hectares and utilizes reverse osmosis technology. It has a daily capacity of 120,000 m³, with 60,000 m³ allocated to Algiers and the remainder to Tipaza. It is situated 20 km from the provincial capital and 35 km west of Algiers. Beni Mered Wastewater Treatment Plant: Situated in Beni Mered commune, Blida province, this plant spans 6 hectares and treats both urban and industrial wastewater. Operational since 2017, it has a nominal capacity of 383,000 PE and a peak flow rate of 51,560 m³/day. The plant is served by two pumping stations with increasing flow rates projected through 2030. Tonic Industry: Established on April 14, 2011, in the Bousmail Industrial Zone of Tipaza province, this plant operates in the paper and packaging sector. Located 20 km from Tipaza and 35 km west of Algiers, it is a key industrial hub within the region. Oued Chiffa: Located in El Hamdania commune, Médéa wilaya, Oued Chiffa flows from the Médéa mountains through the Chiffa gorges and Mitidja plain, emptying into the sea. It has a length of 4 km and is bordered by Djebel Mouzaïa to the north and other mountains to the east and west (Hamaidi et al., 2008). Oued Sebaou: This watershed covers 2,500 km² in the Djurdjura Mountains of northern Algeria, flowing through the Tizi-Ouzou province. The Sébaou River extends approximately 100 km east-southeast of Algiers.

2.2 Study zone and sampling

To enhance the clarity and precision of spatial representation, a detailed map of the study area was constructed using QGIS. This map, accurately displays the various sampling sites and surrounding geographical features, providing a comprehensive visual aid that supports the spatial analysis of environmental data collected during the research. To study the effects of various effluents on Daphnia magna, samples were collected from multiple sources, including desalination and wastewater treatment effluents, Oueds, and industrial watercourses. Sterile plastic bottles were used for sample collection, ensuring contamination-free samples this method was consistently applied across tonic industry, three different samples from various locations within Oued Chiffa and two samples from distinct sites in Oued Sebaou. A visit was made to the desalination station in Fouka, Tipaza, where we were able to collect water samples from tap water intended for citizens in sterile plastic bottles ensuring contamination-free samples. We also collected samples from the wastewater treatment plant provided by the employees. To maintain the water temperature close to its natural state, most of the sample collection was conducted during winter, except for samples from the tonic industry which was in July. This seasonal timing helps preserve the ecological validity of the samples by keeping the water temperature stable. After collection, the samples were immediately transported then brought to the laboratory for use in various bioassays, allowing for the assessment of their effects on Daphnia magna under controlled experimental conditions. This systematic approach ensures reliable and reproducible results in understanding the environmental impacts of different effluents on aquatic life.

2.3 Biological material and experimental Equipment

Behavioral parameters are increasingly recognized as sensitive and early indicators of water quality. For our study, we have chosen Daphnia magna to test its bio-indicator characteristics with respect to pollution in different environments, which reproduces through parthenogenesis. Daphnia, like other cladocerans, play a key role in freshwater ecosystems (Nilssen and Waervagen, 2002). The main idea is to test the tolerance of this species to different types and levels of water pollution. This species was chosen for the study for several reasons it is visible to the naked eye, it reproduces by parthenogenesis, leading to low genetic variability, it has abundant offspring, it is easy to handle and cultivate in the laboratory due to its relatively small size, it has a short life cycle, allowing the observation of pollutant effects over multiple generations, and it is sensitive to a wide range of chemicals (Colbourne, 2011). For the experimental part, we used the following laboratory materials and equipment: Glass aquarium, Pump and oxygen diffuser, Binocular microscope, Inverted microscope. 25 ml test tubes, 200 ml laboratory beaker, spirulina, slides, coverslips, petri dishes, pasteur pipette, incubator and metal spatula.

2.4 Maintenance and cultivation of Daphnia magna in laboratory conditions

We maintained the culture in two large shallow aquariums, filled these aquariums with dechlorinated and oxygenated tap water, ensuring a stable environment for the species. The water quality was carefully monitored, with regular checks to maintain a neutral pH and low hardness (Lari et al., 2018). We kept the temperature between 18°C and 22°C (Olkova, 2020) by using an aquarium heater or submersible heater, given that the experiment at first was during the spring. We also used an air pump to provide regular access to oxygen in the aquarium (Gerritsen et al., 2017). We controlled the lighting to simulate a natural day-night cycle, providing about 12–16 hours of light per day (Krichel et al., 2023). For feeding, we provided a balanced diet of Spirulina, which promotes healthy growth and reproduction, supporting overall vitality (Gerritsen et al., 2017). We ensured not to overfeed, as excess food could degrade water quality. We promptly removed any uneaten food and debris to prevent bacterial growth. Additionally, we performed regular partial water changes of about 10–20% weekly to maintain optimal water conditions (Wagner et al., 2016). By closely monitoring their health and behavior, we could quickly address any issues, such as changes in population dynamics or behavior, indicating potential problems with the environment or food supply. This setup allowed us to maintain a healthy and thriving culture of Daphnia magna for our experiments.

This study employed both chronic and acute toxicity tests to evaluate the effects of various effluents on Daphnia magna. The chronic test, a long-term evaluation, assesses the impact of pollutants over an extended period, typically ranging from several days to several weeks. It focuses on life cycle parameters such as growth, reproduction, and survival to understand the prolonged effects of exposure. This test provides valuable insights into how pollutants affect the organisms over their entire life span. In contrast, the acute test is a short-term assessment conducted over 24 to 48 hours to measure the immediate effects of pollutants. It is designed to determine the rapid toxicity and lethal concentration or dose of a substance, helping to identify the short-term adverse effects on Daphnia magna. During an acute test, feeding is generally not necessary, as the primary purpose is to assess the immediate toxicity and determine the lethal concentration or dose. In our case, we started by exposing young female Daphnia magna, less than 24 hours and from the third brood, to various effluents in the chronic test. These effluents included samples from Oued Chiffa, Oued Sebaou, Tonic industry, Fouka Tipaza desalination plant, Beni Merad wastewater treatment plant. The control group was maintained in dechlorinated tap water. Each treatment, including the control, was replicated four times to ensure statistical reliability. We have also tested four other samples of these effluents with dilution using ultrapure water and distilled water, applying dilution factors of 0.3, 0.5, and 0.7 ) dilution flow helps in dispersing pollutants and reducing their concentrations to acceptable levels (Farhadian et al., 2014). This approach allowed us to evaluate the effects of varying concentrations of the effluents on Daphnia magna. The chronic test setup involved placing the Daphnia individually in 25 ml test tubes. To maintain optimal conditions, the water was aerated continuously using an air pump, and the temperature was controlled using an aquarium heater. The organisms were fed Spirulina, a high-protein microalgae beneficial for their growth and reproduction, every two days. We carefully avoided overfeeding. The culture medium was replaced every three days to maintain clean and stable conditions (Wagner et al., 2016). During the test, juveniles from each brood were collected and counted. Their size was measured from the head to the caudal spine using a micrometer (Ebert, 2022). The following life cycle parameters were recorded:

Table 1

Life History Parameters of *Daphnia magna* During a 21-Day Reproduction Test Measurement.
Parameter	Measurement
Size of Adult Females	Measured in mm at their first reproduction
Size of Juveniles	Measured in mm at first reproduction
Age at Sexual Maturity	The age when Daphnia reach reproductive capability
Age at First Brood	The age at which the first brood of juveniles is produced
Average Brood Interval	The average time in days between successive broods
Brood Size	The average number of offspring per mother at the end of the test
Longevity	The average number of days the mothers remained alive throughout the test
Total Offspring	The average total number of offspring produced by each mother by the end of the test
Number of Broods	The total number of broods produced by each mother during the 21-day test period

We conducted a 48-hour acute test in which Daphnia magna were placed in 200 ml laboratory beakers. During this period, the organisms were not fed, but they were exposed to natural daylight to mimic environmental conditions (Ebert, 2022). This setup allowed us to assess the immediate toxic effects of the effluents on the species under controlled conditions. This approach was used to evaluate the mortality levels, which serve as an indicator of the water’s pollution or purity (Ebert, 2022).

2.4 Statistical analysis

Descriptive Statistics: Summary statistics (mean, median, quartiles, minimum, and maximum) were computed for all variables across the sites. ANOVA and Post-hoc Analysis: ANOVA was performed to test for differences in adult size without dilution across sites, followed by Tukey's post-hoc test to identify pairwise differences. Kruskal-Wallis Test: As a non-parametric alternative to ANOVA, the Kruskal-Wallis test was used to confirm the results. (kruskal,w.h.1952) Principal Component Analysis (PCA): PCA was performed to reduce the dimensionality of the data and identify patterns. Hierarchical Clustering: Clustering was used to group sites based on the similarity of biological metrics. Graphics were generated using R

3.1 Survival rate

The data presented in Table 2 shows the percentage of the survival rate of Daphnia magna at the end of the study across different sites, without dilution. The control group exhibited the highest survival rate at 90%. The survival rate for Daphnia exposed to desalination water was 80%. In the Oued Sebaou sites, site 1 had a survival rate of 70%, while site 2 had a survival rate of 60%. For the Oued Chiffa sites, site 1 showed a survival rate of 70%, site 2 had a survival rate of 50%, and site 3 had a survival rate of 30%. The wastewater treatment plant water resulted in a survival rate of 50%. The tonic industry water had a 0% survival rate. This leads to the conclusion that in desalination water, as well as in Oued Chiffa 1 and Oued Sebaou 1, the survival rates were relatively close to the control, ranging from approximately 70–80%.

Table 2

Percentage of the survival rate of *Daphnia* at the end of the study in the different sites without dilution.
	Survival Rate
Control	90%
Desalination water	80%
Oued sebaou 1	70%
Oued sebaou 2	60%
Oued chiffa 1	70%
Oued chiffa 2	50%
Oued chiffa 3	30%
Wastewater treatment plant water	50%
Tonic industry water	0%

The data presented in Table 3 shows the percentage of the survival rate of Daphnia magna at the end of the study across different sites, with varying dilution factors using ultrapure water. For the control group, the survival rate was 90% at a 0.3 dilution factor, decreasing to 80% at both 0.5 and 0.7 dilution factors. In desalination water, the survival rate was 80% at a 0.3 dilution factor, and 70% at both 0.5 and 0.7 dilution factors. For the Oued Sebaou sites, site 1 showed a survival rate of 70% at a 0.3 dilution factor, increasing to 80% at both 0.5 and 0.7 dilution factors. Site 2 had a survival rate of 60% at a 0.3 dilution factor, and 70% at both 0.5 and 0.7 dilution factors. In the Oued Chiffa sites, site 1 had a survival rate of 70% at a 0.3 dilution factor, which increased to 80% at both 0.5 and 0.7 dilution factors. Site 2 had a survival rate of 50% at a 0.3 dilution factor, and 70% at both 0.5 and 0.7 dilution factors. Site 3 showed a survival rate of 30% at a 0.3 dilution factor, increasing slightly to 40% at both 0.5 and 0.7 dilution factors. For the wastewater, the survival rate remained at 50% at both 0.3 and 0.5 dilution factors, increasing slightly to 60% at a 0.7 dilution factor. The tonic industry water consistently showed a 0% survival rate across all dilution factors. This leads to the conclusion that, with dilution using ultrapure water, survival rates in the control, desalination water, Oued Sebaou 1, and Oued Chiffa 1 remained relatively high and comparable, generally ranging from 70–80%. However, the toxic effect of tonic industry water persisted regardless of dilution.

Table 3

Percentage of the survival rate of daphnia at the end of the study in different sites with dilution using ultrapure water.
	Survival Rate	Survival Rate	Survival Rate
Dilution factors	0.3	0.5	0.7
Control	90%	80%	80%
Desalination water	80%	70%	70%
Oued sebaou 1	70%	80%	80%
Oued sebaou 2	60%	70%	70%
Oued chiffa 1	70%	80%	80%
Oued chiffa 2	50%	70%	70%
Oued chiffa 3	30%	40%	40%
Wastewater treatment plant water	50%	50%	60%
Tonic industry water	0%	0%	0%
3.2 The obtained results without using dilution

The data presented in Fig. 4 shows a metrics without dilution across site presents a comprehensive overview of how various life cycle parameters of Daphnia magna are affected by pollution across different sites without dilution.

The size of adult females at the first brood is highest in the control group and desalination water, ranging from 3.2–3.4 mm. However, this size decreases progressively at the Oued Sebaou and Oued Chiffa sites, reaching as low as 2.3 mm at the most polluted Oued Chiffa site. The wastewater results in a size of 2.4 mm, while no females survived in the tonic industry water.

A similar trend is observed for the size of juveniles at the first brood, with the control group and desalination water having the largest sizes around 1.1–1.2 mm. Juvenile size decreases to as low as 0.3 mm at the most polluted Oued Chiffa site, 0.4 mm in wastewater, and no juveniles survived in tonic industry water.

Longevity is maximized at 20 days in the control group, declining to 14 days at the most polluted Oued Chiffa sites and wastewater, with no survival in tonic industry water.

The brood interval remains stable at 3 days in the control, desalination water, and lightly polluted Oued Sebaou 1 and Oued Chiffa 1 sites. It increases to 4–5 days at more polluted sites, reaching 4 days in wastewater. No broods were produced in tonic industry water.

Age at first brood follows a similar pattern, staying around 7.5-8 days in the control and desalination, then progressively increasing to 10.5 days in wastewater. No first broods were recorded in tonic industry water.

Brood size decreases from 10 eggs in the control and desalination to only 3–4 eggs at the most polluted Oued Chiffa sites and wastewater. No broods were produced in tonic industry water.

The number of egg-laying events per female drops from 4.7 in the control to only 1 at the most polluted sites. No egg-laying occurred in tonic industry water.

The total number of offspring per female drastically declines from 42 in the control to only 3–8 at polluted Oued Chiffa sites and wastewater. No offspring were produced in tonic industry water.

Sizes of females and juveniles at the second brood are generally larger than the first brood, reaching 3.4 mm in the control and desalination. However, the most polluted sites did not allow Daphnia to reach a second brood.

Sex ratio without dilution

The data shows the percentage of malformation and the percentage of males in the offspring of Daphnia magna during the 21-day test across different effluents (Table 4). The control group exhibited no malformations and no males in the offspring. Similarly, for desalination water and Oued Sebaou site 1 also showed 0% malformation and 0% male offspring. However, Oued Sebaou site 2 recorded a slight presence of males at 1% with no malformations. In Oued Chiffa sites, site 1 had 0% malformation and no male offspring, while site 2 showed a significant increase in male percentage at 12%, still with no malformation. The most extreme results were observed at Oued Chiffa site 3 and the wastewater treatment plant water, both showing high malformation rates of 25% and 15% respectively, along with a striking 90% male offspring in both cases. When it comes to tonic industry water no results were recorded.

Table 4

Embryo-toxicity and percentage of males in the Offspring of *Daphnia magna* during the 21 days test exposed to different effluents without dilution
	Malformation %	Male %
Control	0%	0%
Desalination water	0%	0%
Oued sebaou 1	0%	0%
Oued sebaou 2	0%	1%
Oued chiffa 1	0%	0%
Oued chiffa 2	0%	12%
Oued chiffa 3	25%	90%
Wastewater treatment plant water	15%	90%
Tonic industry water	/	/

3.3 The obtained results while using ultrapure water for dilution

The data presented in Fig. 5 shows a metrics with dilution using ultrapure water across site presents a comprehensive overview of how various life cycle parameters of Daphnia magna affected by pollution across different sites incorporating average results between dilution factors (0.3, 0.5, and 0.7).

The size of adult females at the first brood is highest in the control group and desalination water, ranging from 3.3 mm to 3.5 mm. However, this size decreases progressively at the Oued Sebaou and Oued Chiffa sites, reaching as low as 2.5 mm at site 2 of Oued Sebaou and 2.6 mm at the most polluted Oued Chiffa site. The wastewater treatment plant water results in an adult female size of 2.8 mm, while no females survived in the tonic industry water.

A similar trend is observed for the size of juveniles at the first brood, with the control group and desalination water having the largest sizes around 1.3 mm. Juvenile size decreases to as low as 0.89 mm at site 2 of Oued Sebaou and 0.5 mm at the most polluted Oued Chiffa site. The wastewater treatment plant water results in a juvenile size of 0.6 mm, with no juveniles surviving in tonic industry water.

Longevity is maximized at 20 days in the control group, slightly declining to 19 days in desalination water. At the Oued Sebaou sites, longevity decreases to 17.9 days at site 1 and 17 days at site 2. In the more polluted Oued Chiffa sites, longevity further declines to 17.9 days, 15 days, and 14 days for sites 1, 2, and 3, respectively. The wastewater treatment plant water results in a longevity of 14 days, with no survival recorded for Daphnia magna exposed to tonic industry water.

The brood interval remains stable at 2 days in the control and desalination water. At the Oued Sebaou sites, the brood interval increases to 3 days at both sites. In the Oued Chiffa sites, the intervals range from 3 days to 4 days, with the wastewater treatment plant water also resulting in a 4-day interval. No brood intervals were recorded for Daphnia magna exposed to tonic industry water.

Age at first brood follows a similar pattern, remaining at 7.5 days in the control and desalination water. At the Oued Sebaou sites, the age at first brood release is 7 days at site 1 and 9 days at site 2. In the Oued Chiffa sites, the ages increase to 8.2 days, 8.8 days, and 10 days for sites 1, 2, and 3, respectively. The wastewater treatment plant water results in an age at first brood release of 9.9 days, with no measurable age recorded for Daphnia magna exposed to tonic industry water.

Brood size decreases from 12 eggs in the control group to 11 eggs in desalination water. At the Oued Sebaou sites, the brood size is 9.9 at site 1 and 8.7 at site 2. In the Oued Chiffa sites, brood sizes decline further to 9, 5.9, and 4 for sites 1, 2, and 3, respectively. The wastewater result in a brood size of 5.2, with no brood size recorded for Daphnia magna exposed to tonic industry water

The number of egg-laying events per female drops from 5 in the control group to only 1 at the most polluted Oued Chiffa sites and wastewater treatment plant. it No egg-laying occurred in tonic industry water. At Oued Sebaou sites, was 4.4 at site 1 and 4 at site 2. In the Oued Chiffa sites, the numbers were 4, 2 for sites 1, 2 respectively.

The total number of offspring per female drastically declines from 44 in the control group to 40 in desalination water. At the Oued Sebaou sites, the number of offspring per female was 36 at site 1 and 28 at site 2. At the Oued Sebaou sites, the number of offspring per female was 36 at site 1 and 28 at site 2. In the Oued Chiffa sites, the offspring counts were 31, 8, and 3 for sites 1, 2, and 3, respectively. The wastewater treatment plant water resulted in 3 offspring per female. No offspring were produced in tonic industry water.

The control group had an adult female size of 3.4 mm. In desalination water, the size was slightly larger at 3.6 mm. At the Oued Sebaou sites, adult females measured 3.4 mm at site 1 and 2.7 mm at site 2. In the Oued Chiffa sites, the sizes were 3.5 mm and 3.1 mm for sites 1 and 2, respectively, while no measurable size was recorded for site 3. Similarly, no sizes were recorded for females exposed to wastewater treatment plant water and tonic industry water.

The control group had juvenile sizes of 1.4 mm. In desalination water, the size remained at 1.4 mm. At the Oued Sebaou sites, juvenile sizes were 1.2 mm at site 1 and 1 mm at site 2. The Oued Chiffa sites recorded juvenile sizes of 1.2 mm at site 1 and 0.86 mm at site 2, with no measurable size recorded for site 3. Similarly, no sizes were recorded for juveniles exposed to wastewater treatment plant water and tonic industry water.

Sex ratio with a dilution

The data presented in Table 5 shows the percentage of malformations and the percentage of males in the offspring of Daphnia magna during a 21-day test across different effluents with a dilution factor of 0.3, 0.5, and 0.7. The control group, desalination water, and Oued Sebaou sites 1 and 2 exhibited no malformations and no males in the offspring. Oued Chiffa site 1 also recorded 0% malformation and 0% male offspring. At Oued Chiffa site 2, there was a 6% occurrence of male offspring, still with no malformation. The most significant effects were observed at Oued Chiffa site 3 and in the wastewater treatment plant water, with malformation rates of 15% and 10%, respectively, and a striking 70% male offspring in both cases. No results were recorded for the tonic industry water in terms of malformation or male offspring percentage.

Table 5

Embryo-toxicity and percentage of males in the Offspring of *Daphnia magna* during the 21 days test exposed to different effluents with dilution.
	Malformation %	Male %
Control	0%	0%
Desalination water	0%	0%
Oued sebaou 1	0%	0%
Oued sebaou 2	0%	0%
Oued chiffa 1	0%	0%
Oued chiffa 2	0%	6%
Oued chiffa 3	15%	70%
Wastewater treatment plant water	10%	70%
Tonic industry water	/	/

3.4 The obtained results while using distilled water for dilution

In a parallel experiment, the same procedure was conducted using distilled water instead of ultrapure water. In this case, the results indicated a complete mortality of all Daphnia magna specimens across various dilutions (0.3, 0.5, and 0.7) and different effluents (desalination water, oued sebaou, oued chiffa, wastewater treatment plant water, tonic industry) in less than 36 hours.

3.5 The obtained results in an acute test

The acute test obtained results are interpreted according to the following factor, in our acute test, we focused exclusively on the mortality of Daphnia magna as the primary endpoint. The results obtained over the 48-hour exposure period are presented in the following table, highlighting the mortality rates observed of Daphnia magna exposed to different water effluents.

The data presented in the Table 6 shows the mortality percentage of Daphnia magna during a 48-hour acute test across different water effluents. The control group, desalination water, and Oued Sebaou sites 1 and 2 exhibited minimal mortality, with only 2% of Daphnia affected by the 48-hour mark, indicating low toxicity levels. Oued Chiffa site 1 also recorded low mortality rates, with 2% observed by 48 hours. However, Oued Chiffa site 2 showed a higher impact, with mortality reaching 20% at the 36-hour and 48-hour marks. The most significant effects were observed at Oued Chiffa site 3 and the wastewater treatment plant water, where mortality rates reached 70% and 60%, respectively, by the end of the test. The tonic industry water was the most toxic, resulting in 100% mortality within 36 hours.

Table 6

*Daphnia magna* mortality rate within 48 hours
Time	12h	24h	36h	48h
Control	0%	0%	0%	2%
Desalination water	0%	0%	2%	2%
Oued sebaou 1	0%	0%	2%	2%
Oued sebaou 2	0%	5%	15%	15%
Oued chiffa 1	0%0	0%	2%	2%
Oued chiffa 2	0%	10%	20%	20%
Oued chiffa 3	15%	30%	50%	70%
Wastewater treatment plant water	15%	20%	60%	60%
Tonic industry water	50%	80%	100%	100%

3.6 combination between result with and without dilution

According to Fig. 6, after comparing the results of Daphnia magna life cycle parameters with and without dilution, it is evident that the life cycle is less affected while using dilution. The data indicates that higher dilution factors lead to improved reproductive outcomes and overall vitality in Daphnia magna. This observation supports the conclusion that cleaner water conditions enhance the resilience and reproductive success of these organisms, highlighting the importance of water quality in aquatic ecosystems.

This study provides valuable insights to understanding how different effluents affect Daphnia magna, which is critical for monitoring and managing aquatic ecosystems. By examining the effects of effluents from various sources—Oued Chiffa, Oued Sebaou, Tonic Industry, Fouka Tipaza Desalination Plant, and Beni Merad Wastewater Treatment Plant—on the life history parameters of Daphnia magna. The impacts on life history parameters such as reproduction, growth, and survival rates of Daphnia magna are particularly important. These parameters can serve as indicators of the overall health of aquatic ecosystems, and changes in them might reflect broader environmental changes or stressors. These findings can thus inform better regulatory practices and pollution management strategies to protect aquatic environments.

The size of adult females at their first brood shows variation compared to the control group, where females exhibit normal sizes. In desalination water, females are slightly larger than those in the control, indicating minimal or no pollution. At Oued Sebaou Site 1 and Oued Chiffa Site 1, the size is smaller yet close to the control group, suggesting that these sites are not polluted. However, at Oued Chiffa Site 2 and Oued Sebaou Site 2, the slightly smaller size of females, which is also under 2.5 mm, indicates a slight presence of pollution. More concerning are the females exposed to wastewater and Oued Chiffa Site 3, where a significant reduction in size points to severe pollution. The most extreme case is the Tonic Industry site, where no values were recorded due to the death of all Daphnia on the first day, indicating the highest level of pollution. These observations suggest that water quality directly impacts female size, with more polluted conditions leading to a noticeable reduction. The decrease in size is associated with accelerated maturity, consistent with findings from Lampert and Wolf (1986), Touati and Samraoui (2002), Chakri (2007), and Manar (2008).

The size of juvenile Daphnia at their first brood shows variation compared to the control group, where juveniles exhibit normal sizes. In desalination water, juveniles are slightly larger than those in the control, indicating minimal or no pollution. At Oued Sebaou Site 1 and Oued Chiffa Site 1, the size is smaller yet close to the control group, suggesting that these sites are not significantly polluted. However, at Oued Chiffa Site 2 and Oued Sebaou Site 2, the slightly smaller size of juveniles, which is also under 0.7 mm, indicates a slight presence of pollution. More concerning are the juveniles exposed to wastewater and Oued Chiffa Site 3, where a significant reduction in size points to severe pollution. The most extreme case is the Tonic Industry site, where no values were recorded due to its death on the first day, indicating the highest level of pollution. Accelerating maturity while reducing size provides an advantage for Daphnia, allowing it to reproduce before reaching a size where it becomes more vulnerable to predators. Therefore, the reduction in size is an adaptive life-history strategy of Daphnia magna that enhances its survival in environments with pollutants or effluents.

The longevity of Daphnia decreases with increasing pollution levels. While the control group has a lifespan of 20 days, desalination water and sites like Oued Sebaou Site 1 and Oued Chiffa Site 1 show minimal reductions. In contrast, longevity drops to 16 days at Oued Chiffa Site 2 and 14 days at wastewater and Oued Chiffa Site 3, indicating more severe pollution. The Tonic Industry site has no recorded longevity due to the death of all Daphnia, reflecting the highest pollution level. This trend of reduced lifespan in polluted conditions is consistent with previous studies (Bougueffa and Boutalbi, 2008; Kortez et al., 2008; Benghorieb and Siline, 2012).

The brood interval of Daphnia is generally 3 days in the control group and in desalination water and at Oued Sebaou Site 1, indicating minimal or no pollution. It increases to 4 days at Oued Sebaou Site 2 and Oued Chiffa Site 3, suggesting slight pollution. The interval extends to 5 days at Oued Chiffa Site 2 and wastewater treatment plant water, indicating more severe pollution. The Tonic Industry site shows no recorded data due to the death of all Daphnia, reflecting the highest pollution level. Overall, a longer brood interval is associated with higher pollution levels. This interval is almost the same compared to the studies by Touati and Samraoui (2002) and Chakri (2007), where it was only 2–4 days. Except for two sites it is probably due to the fact that intrinsic toxicity of the effluent disrupts Daphnia reproduction by acting as an endocrine disruptor, interfering with hormonal balance. Additionally, the overload on their detoxification systems forces Daphnia to prioritize survival over reproduction, resulting in longer brood intervals. Similar effects were reported by Bougueffa and Boutalbi (2008), who found that hospital effluents caused a significant increase in brood intervals of Daphnia magna to 6–8 days.

The age at first brood release for Daphnia is similar in all of the control group and desalination water oued chiffa site 1 and oued sebaou site 1, indicating minimal or no pollution. It increases in Oued Sebaou Site 2 and Oued Chiffa Site 3, suggesting moderate to severe pollution. The Tonic Industry site shows no recorded data due to the death of all Daphnia, reflecting the highest pollution level. Overall, a longer age at first brood release is associated with higher pollution levels. These results are entirely similar to those of Touati & Samraoui (2002), Chakri (2007), Bougueffa & Boutalbi (2008), and Benghorieb & Siline (2012).

The brood size of Daphnia is the same across the control group, desalination water, and Oued Sebaou Site 1, indicating minimal or no pollution. It decreases at Oued Sebaou Site 2 and Oued Chiffa Site 1, suggesting slight pollution. The brood size is further reduced at Oued Chiffa Sites 2 and 3, and wastewater treatment plant water, indicating moderate to severe pollution. The Tonic Industry site shows no recorded data due to the death of all Daphnia, reflecting the highest pollution level. Overall, a smaller brood size is associated with higher pollution levels. These findings are consistent with studies by Allan & Goulden (1980) and Daniels & Allan (1981).

The control group exhibited the highest number of egg-laying events per female at 4.7, indicating a healthy reproductive activity under optimal conditions. Desalination water, Oued Sebaou Site 1, and Oued Chiffa Site 1 showed a slightly lower but still robust number of egg-laying events, at 4 events each, suggesting minimal environmental stress and pollution. The reproductive activity begins to decline at Oued Sebaou Site 2, with more significant reduction in Oued Chiffa Site 2 and Oued Chiffa Site 3, reflecting increased pollution and its detrimental effects on reproduction. The most severe impact is seen at the wastewater treatment plant, where the number of egg-laying events drops to 1 highlighting the extreme toxicity of these environments on reproductive functions.

The number of offspring per female varies significantly across different sites, with the control group recording the highest, indicating optimal conditions. Desalination water and Oued Sebaou Site 1 follow closely, showing minimal stress. However, offspring numbers decline at Oued Sebaou Site 2 and Oued Chiffa Site 1, suggesting moderate pollution. A remarkable reduction is observed at Oued Chiffa Sites 2 and 3, where offspring numbers drop sharply, reflecting severe pollution. The lowest reproductive success is observed at the wastewater treatment plant and Tonic industry water sites, where pollution severely disrupts reproductive processes. Similar findings were reported by Touati & Samraoui (2002).

The size of females and juveniles at the second reproduction is greater than at the first (Touati and Samraoui, 2002; Chakri, 2007). These findings are also supported by a study by Hall and Burns (2003). The analysis of the table shows that the presence of males is notably absent in control, desalination water, and several Oued Sebaou samples, while it is significantly high in Oued Chiffa 3 and wastewater treatment plant water, indicating possible environmental factors that influence sex ratios. Malformations are minimal in most samples but increase substantially in Oued Chiffa 3 and wastewater treatment plant water, suggesting higher pollutant levels leading to developmental issues. This aligns with findings from Hansson et al. (2007), which demonstrate that pollutants can disrupt reproductive health and development in Daphnia magna.

In this study, we observed that the results from the experiment with varying dilution factors using ultrapure water differed slightly from those obtained without dilution, the outcomes showed less pronounced effects on Daphnia magna compared to the undiluted samples. This variation can be attributed to the dilution of pollutants, which reduces their concentration and, consequently, their impact on the organisms. By decreasing the pollutant concentration through dilution, we effectively mitigated some of the stressors affecting Daphnia magna, leading to less severe responses in growth, development, and reproductive metrics. This demonstrates how dilution with ultrapure water can alter the perceived impact of pollutants on aquatic organisms.

The total death of our Daphnia magna specimens when using distilled water is likely due to osmotic shock caused by the lack of essential ions and minerals needed to maintain osmotic balance. Distilled water, devoid of these critical components, can disrupt the physiological processes of aquatic organisms, leading to rapid mortality. Also the absence of necessary nutrients and potential water quality issues might have further contributed to the adverse conditions, resulting its death, it is also possible that the distilled water was of poor quality or contaminated, which could have contributed to the rapid death of the Daphnia magna. If the distilled water was not properly purified or handled, it might have contained harmful substances or lacked the necessary stability to support aquatic life. This could lead to adverse effects on the organisms, including rapid mortality.

The results of the acute test indicate that Daphnia magna exhibits minimal mortality in control and desalination water, with only slight increases over time. Similarly, Oued Sebaou 1 and Oued Chiffa 1 show low mortality rates, suggesting relatively low toxicity. Conversely, Oued Sebaou 2 and Oued Chiffa 2 demonstrate moderate toxicity, with mortality rates increasing progressively. Oued Chiffa 3 and wastewater treatment plant water are highly toxic, causing substantial mortality, which escalates rapidly. Tonic industry water is extremely toxic, resulting in complete mortality of Daphnia magna within 48 hours. These findings reflect significant variations in toxicity levels among the water samples, with some exhibiting severe adverse effects on aquatic organisms.

The results indicate that as the dilution factor increases, the overall health and reproductive success of Daphnia magna improve, confirming our hypothesis that cleaner water yields better outcomes. This trend suggests that lower concentrations of pollutants enhance the life cycle parameters of these organisms. Studies by (Baker et al., 2003) support this theory, demonstrating that reduced pollutant levels in aquatic environments lead to increased reproductive performance and overall vitality in freshwater species. By comparing the results from the three different dilution factors used, we can conclude that higher dilution correlates with improved biological metrics, reinforcing the importance of water quality in sustaining aquatic life.

All life cycle parameters of Daphnia magna are negatively impacted by pollution, with progressive reductions in size, longevity, reproduction and survival. The most polluted sites were completely lethal. These results highlight the value of D. magna as a sensitive bioindicator of water quality. The map below illustrates the variation in pollution levels across all the site

4.1 The Importance of the Study

This study is indeed crucial, as it employs Daphnia magna—a well-established bio-indicator species—to evaluate pollution levels across various water sources, including rivers, industrial effluents, wastewater, and desalination water. By examining how pollutants impact the life history traits and survival of Daphnia magna, we gain early insights into the presence of harmful substances in these water bodies, thus enhancing our environmental monitoring capabilities. The implications of this research extend beyond immediate water quality concerns. Understanding and mitigating pollution in these smaller water systems is crucial because contaminants can accumulate and be transported downstream, eventually affecting larger bodies of waters. This, in turn, impacts the broader aquatic ecosystems and the rich biodiversity they support. Therefore, this study contributes to both the protection and conservation of freshwater environments and the health of marine ecosystems.

4.2 The Impact of the study on the Oceans and Aquatic Ecosystems

The health of freshwater ecosystems is intrinsically linked to the well-being of oceans and other aquatic environments. Pollutants detected in rivers and streams can travel through watersheds, reaching larger water bodies, including estuaries and oceans. This study's focus on detecting pollution using Daphnia magna helps identify these contaminants before they reach larger aquatic systems, where their effects can be magnified. Polluted waters can lead to the degradation of marine habitats, such as coral reefs and coastal ecosystems, disrupting the delicate balance of marine life and reducing biodiversity. By conducting this study, we contribute to efforts aimed at safeguarding these ecosystems, ensuring that pollutants are identified and managed before they cause irreparable harm. The insights gained from this research can inform policies and strategies for pollution control, ultimately contributing to the preservation of the oceans and the myriad species they harbor.

4.3 Relevance to Human Health

The relationship between water quality and human health is direct, particularly when considering the use of desalination water. Desalination plants are becoming an increasingly vital source of potable water, especially in arid regions, such Algeria. However, the quality of desalination water must monitored to ensure it is free from harmful contaminants that could pose risks to human health. This study, by examining the effects of desalination water on Daphnia magna, provides a proxy for understanding potential risks to human health. Pollutants that adversely affect Daphnia may also have harmful effects on humans, particularly through exposure to toxic compounds or pathogens that can bypass conventional water treatment processes. Furthermore, pollutants that enter aquatic environments can accumulate in the tissues of aquatic species, such as fish and shellfish, which are later consumed by humans. The bioaccumulation of these harmful substances poses a significant risk, leading to potential health issues, including toxicity and long-term exposure effects. By identifying and mitigating these pollutants through our study, we can prevent their accumulation in aquatic food chains, ultimately protecting human health. Also, this research plays a pivotal role in safeguarding public health by ensuring that the water we consume is clean and safe.

4.4 Implications for Aquaculture

Aquaculture relies heavily on the availability of clean water to maintain healthy stocks of fish and other aquatic organisms. This study is directly relevant to aquaculture because it helps identify water sources that are safe for use, ensuring that the water used in fish farms is free from harmful pollutants. By demonstrating that certain water sources are clean and suitable for aquaculture, this research can help expand the industry sustainably. Moreover, the potential for using treated wastewater in aquaculture presents a promising avenue for reducing water scarcity and promoting eco-friendly practices. After thorough treatment and testing, wastewater could be repurposed for aquaculture, offering an economical and environmentally sustainable solution. By recycling wastewater in this way, we not only conserve freshwater resources but also reduce the environmental footprint of aquaculture, contributing to a more sustainable and circular economy. This approach could revolutionize the aquaculture industry, making it more resilient and adaptable to the challenges posed by water scarcity and pollution.

4.6 Enhancing Water Pollution Analysis with AI

Building on this study, a forthcoming article will explore innovative approaches by integrating AI technologies to enhance the analysis of water pollution impacts on Daphnia magna. This research will leverage advanced data processing techniques to provide more precise and actionable insights for environmental monitoring.

Understanding the mechanisms by which pollutants affect Daphnia is crucial for using them as bio-indicators in environmental monitoring. By studying the specific physiological, behavioral, reproductive, and molecular responses to different types of pollutants, researchers can develop more accurate and sensitive methods for detecting and assessing the impact of environmental contaminants. This knowledge can also inform the development of regulatory policies and strategies for mitigating the effects of pollution on aquatic ecosystems. (Lampert, 2011). The study has investigated the impact of various water effluents on Daphnia magna, focusing on survival rates, reproductive success, and overall health. Tonic Industry Water exhibited the highest toxicity levels, significantly affecting the survival and reproductive rates of Daphnia magna. The effluent was classified as extremely dangerous to aquatic life. Oued Chiffa Site 3 and Wastewater Treatment Plant Water Both sites were found to present severe pollution. Daphnia magna exposed to these waters showed markedly reduced survival rates and reproductive output, indicating high levels of contamination. Oued Chiffa Site 2 and Oued Sebaou 2 these sites displayed moderate pollution levels, with some adverse effects on Daphnia magna, though they were less severe compared to the aforementioned sites. Oued Chiffa Site 1, Oued Sebaou Site 1, and Desalination Water these water sources were relatively clean, with minimal impacts on the species. The research highlights the significant variability in water quality and its impact on Daphnia magna. Tonic industry effluents pose severe risks to aquatic ecosystems, while wastewater from Oued Chiffa Site 3 and other treatment facilities also indicates high pollution levels. In contrast, cleaner sites such as Oued Chiffa Site 1 and Oued Sebaou Site 1 are relatively safer. These findings underscore the utility of Daphnia magna as a bio-indicator for assessing environmental pollution and provide valuable insights into the specific pollution levels of different water sources. Several limitations were identified in this study such as: Geographic Limitations the study was confined to specific sites, which may not fully represent the variability of pollution across different regions. Sampling Frequency limited sampling periods may not capture temporal variations in pollution levels and their effects on Daphnia magna. Dilution Factors while dilution factors were considered, they may not fully account for real-world scenarios where effluent concentrations vary.

Suggestions for Future Research: Extended geographic scope future studies should include a broader range of sites to better understand regional variations in pollution levels and their effects on bio-indicators. Long-Term monitoring it could provide more comprehensive data on the temporal effects of pollution on aquatic life. Assessment of Additional Pollutants by expanding the study to include a wider range of pollutants could offer a more detailed understanding of their combined effects on Daphnia magna and other bio-indicator species. Mitigation strategies research into potential mitigation measures for reducing pollution from high-risk sources such as the tonic industry and wastewater treatment facilities could help in developing effective environmental protection strategies.

We propose the following perspectives: Develop and upgrade wastewater treatment plants to better manage and reduce pollutants.Implement mandatory yearly reports to transparently assess and address environmental conditions. Establish a system for the regular review and updating of environmental policies and practices.Create dedicated laboratories for in-depth environmental monitoring and research. Finally, this study should be further developed to enhance ecosystem health monitoring and safeguard biodiversity, which is crucial for maintaining ecological balance.

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Data Availability Statement.

The data used in this work are results derived from experiments conducted as part of this research in the Laboratory for the Conservation and Valorization of Marine Resources (LCVRM) at ENSSMAL.

Conflict of Interest: The authors declare that they have no conflict of interest

No competing interests reported.

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Testing resistance of Daphnia magna in various environments

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