Water Monitoring Practices 2.0—Water Fleas as Key Species in Ecotoxicology and Risk Assessment

Abstract

Humanity faces the great challenges arising from pollution and climate change which evidently lead to the irreversible effects observed on the planet. It is now more important than ever to monitor and safeguard the ecosystem as it has been highlighted by governments and scientists. Conventional approaches for water pollution rely on the detection of chemicals in the environment. However, these descriptive observations when compared against water quality standards used as metrics for pollution are unable to predict pollution early or capture the extent of its impact. This weakness is reflected in the legislation and the thresholds for emerging pollutants such as pharmaceuticals and nanomaterials. To bridge the gap and to understand the underlying mechanisms for toxicity, research in the field of molecular ecotoxicology shifts more and more towards the integration of model systems, in silico approaches and molecular information as endpoints. Focusing on the freshwater ecosystem, daphnids are key species employed in risk assessment which are characterised as highly responsive to pollutants and physical stressors. The translation of molecular information describing the physiology of these organisms provides novel and sensitive metrics for pollution assessment.

1. Introduction to the Challenges and Consequences of Freshwater Pollution

Freshwater pollution has become a topic of great environmental concern in recent years, with the disruptions in the delicate equilibrium of aquatic ecosystems while jeopardizing human health. Environmental pollution has surpassed disease as the leading cause of death worldwide. According to the European Commission, pollution is responsible for the deaths of 9 million people per year [1], with 1.8 million deaths being caused by aquatic pollution alone, while the poor quality of drinking water is the leading cause of death, responsible for over 50 different diseases and 50% of child deaths worldwide [2].

Anthropogenic sources such as industrial effluents, agricultural waste and urban waste significantly impact freshwater ecosystems, primarily by the presence of emerging contaminants (ECs). These pollutants include pharmaceutically active compounds (PhACs), pesticides, industrial and household chemicals, personal care products (PCPs), metals, and industrial additives, and pose a serious threat to aquatic environments. These compounds are characterised by their high chemical stability, persistent byproducts and toxic effects on the health of humans and the ecosystems even at very low concentrations ranging from ng L⁻¹ to μg L⁻¹ (

Table 1. Environmentally relevant concentrations of pharmaceuticals.

Category	Pharmaceutical	Concentration in the Environment (ng/L)	Reference Location
Antibiotic	Amoxicillin	1.15	[10] Turkey
	Trimethoprim	45	[10] France
	Erythromycin	99.22	[10] Turkey
	Azithromycin	147.28	[10] USA
Anticancer	Ifosfamide	41	[11] Spain
	Methotrexate	53	[12] Canada
	Cyclophosphamide	500	[11] Taiwan
Antidepressant	Fluoxetine	43.6	[13] Hong Kong
	Venlafaxine	49.2	[13] Scotland
	Sertraline	335.7	[13] Hong Kong
Antidiabetic	Gliclazide	75	[14] South Africa
	Irbesartan	759	[14] South Africa
	Metformin	41,000	[15] Canada
Antiepileptic	Lamotrigine	108	[16] USA
	Carbamazepine	330	[16] USA
	Gabapentin	2790	[16] USA
β-blocker	Propranolol	1900	[17] USA
	Atenolol	3800	[17] Hong Kong
NSAIDs	Indomethacin	100	[18] South Korea
	Ibuprofen	5300	[19] Poland
	Diclofenac	9000	[19] Poland
	Acetyl salicylic acid	440	[20] Spain
Estrogens	ethinyl estradiol	2.58	[21] Iran
	Estrone	51.7	[22] China
	β-estradiol	107	[23] South Africa

). Several pollutants, particularly pharmaceuticals such as paracetamol and ibuprofen, were recorded at their highest levels in the effluents of low income countries because of their poor water sanitation and lack of wastewater infrastructures [3]. ECs enter freshwater systems through various pathways, such as the surface runoff from agricultural or urban areas, the direct discharge from wastewater treatment plants (WWTPs) of municipal, hospital or industrial waste, and landfill leachate [4]. Their main ecological concerns include the endocrine disruptive disorders, damage to the immune system of aquatic organisms and potential microbiological resistance [5]. Phthalate esters (PAEs) and bisphenol-A (BPA) are the main endocrine disrupting chemicals (EDCs) present in drinking water [6], mimicking hormones in various aquatic species, with mollusks, crustaceans and amphibians showing particular sensitivity [7]; Malathion is a widely used organophosphorus insecticide which is very toxic to fish and other invertebrates that harms the melanomacrophage and hematopoietic centres of several fish organs and causes tissue damage to their liver and gills at very low concentrations [8]; tetracycline is a predominant antibiotic in aquatic environments as it cannot be removed by WWTPs, and has been proven to be highly toxic to fish, crustaceans and insects while also inducing the emergence of antibiotic-resistant genes into the environment that could be passed to freshwater species [9].

There are several ways in which anthropogenic sources damage aquatic ecosystems. The use and release of nitrogenous or phosphate fertilizers in the environment has drastic effects on freshwater habitats [24]. The accumulation of nutrients such as nitrogen and phosphorus in the water body causes the proliferation of algae and a consequent decrease in oxygen levels in a phenomenon known as eutrophication, that constitutes a severe threat to aquatic organisms. On the other hand, microplastics have also become a serious environmental problem, as large volumes of plastic debris originating from different synthetic polymeric materials have reached freshwater environments. This endangers aquatic life through bodily injury, decreased reproductive rate and increased mortality, especially in fish [24]. Additionally, heavy metals (mainly mercury, lead and cadmium) also represent a rising concern because of their abundance in water bodies and their potential to bioaccumulate, as they are not easily excreted and tend to accumulate in the fat tissue of fish and amphibians, leading to biomagnification at higher trophic levels.

Given the presence and importance of aquatic environments, and freshwater especially, safeguarding them is more essential and pressing. Freshwater ecosystems play a crucial role in nutrient and salt exchange between aquatic and terrestrial habitats while also serving as vital links between humans and essential resources. Human civilization developed based on water use, and biological resources found in freshwater have always constituted fundamental sources of food. Especially now, as water sustainability becomes a growing concern for many nations, water supplies should be self-sufficient and capable of meeting diverse needs, including agriculture, municipal use, and industry [25]. Water supplies should also remain consistent, despite climate change impacts, i.e., the lack of rainfall and drought. The presence of water is crucial since it interconnects different environments by the dissolution of nutrients originating from plant material or animal products and transporting them back into the soil where they can be recycled. However, freshwater ecosystems and their biota are under severe threat, with species populations declining by 84%; a significantly higher rate than in other ecosystems [26].

Conventionally overlooked compared to estuarine and coastal pollution due to its complexity and reduced public visibility, freshwater pollution must now be thoroughly assessed to mitigate its harmful effects on both the environment and human health, while also addressing the delayed policy actions and existing data gaps concerning freshwater contaminants. Several pollutants already investigated in the early 2000s in marine environments, such as microplastics, have only recently received scientific recognition in freshwater ecosystems, and therefore now require updated methods to assess their impact at environmentally relevant concentrations [27]. Traditional approaches to measuring aquatic pollution have primarily focused on specific anthropogenic chemical compounds, providing insights into water quality and safety by assessing its physical, chemical, and biological properties.

This review focused on daphnids as sentinel species used for the evaluation of the biological effects of two increasingly significant classes of emerging contaminants: pharmaceuticals and nanomaterials. While literature often addresses these pollutants independently or in marine contexts, in this review, we emphasized their sublethal and long-term effects in freshwater environments, where detection remains difficult and ecological impacts are underexplored. With the aim to highlight the predictive value and sensitivity to low-dose exposures of daphnids, this comprehensive analysis aims to bridge current gaps, propose effect-based monitoring approaches as early warning systems in ecotoxicology, and an advancement in risk assessment using this well established freshwater model system.

The review was developed through a structured search and critical evaluation of scientific literature related to the ecotoxicological effects of pharmaceuticals and nanomaterials on daphnids. With the relevant studies identified using academic databases such as PubMed, Web of Science, and Scopus, and our searches spanning from 2018 to 2024 as possible. Research studies were included based on their focus on freshwater environments, the use of daphnids as model organisms, and the assessment of sublethal, molecular, or physiological endpoints following exposure to pharmaceuticals or nanomaterials. Preference was given to peer-reviewed articles reporting environmentally relevant concentrations and studies contributing to the understanding of mechanisms of toxicity. Studies conducted in marine environments, lacking direct experimental work with daphnids, or non-English publications, and non-peer-reviewed sources were excluded. The selection process ensured that only high-quality, relevant literature was considered, providing a strong basis to capture the potential of daphnids in monitoring and risk assessment of emerging contaminants.

2. Revolutionizing Risk Assessment with Novel Approach Methodologies

2.1. Limitations of the Current Approaches in Risk Assessment

It is evident from the above that the existing water monitoring practices frequently fall short in their realism and fail to provide mechanistic insights which are essential for accurate ecotoxicological risk assessment. Water and sediment quality are usually assessed through targeted analytical methods that focus on the spectrum of known chemicals of concern [28]. The EU Water Framework Directive and the Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH) regulation (EC 1907/2006) which came into effect in June 2007 [29] and many other regulations have contributed to the development of the world’s most extensive database. Despite this, crucial data are generated with research studies on specific pollutants. However, much molecular information is required for a thorough and reliable risk assessment which remains incomplete or missing for many of the thousands of chemicals available on the European market [30]. Therefore, completing the REACH database in line with the current information requirements outlined in Annexes VII to XI would likely require over a decade and significant testing resources. The duration and cost of traditional animal testing make it impractical to conduct for the vast number of chemicals currently available on the market and those expected to be introduced in the coming years. In addition, target analytes represent only a small fraction of the total chemical content present in the environment and are selected based on prior knowledge of their occurrence in specific contexts. As a result, targeted analysis does not account for unlisted chemicals or transformation products that may form through biotic and abiotic processes [31]. The effects of chemical mixtures are also another aspect in which the existing animal-centred approach fails to offer sufficient solutions [30]. Furthermore, a comprehensive evaluation of mixture effects requires systematic data collection for individual chemicals, documenting hazard metrics associated with specific effects, target organs, mechanisms of action, adverse outcome pathways (AOPs), and related characteristics [32]. However, traditional in vivo studies typically do not provide such detailed mechanistic insights, introducing uncertainty into the assessment. Additionally, testing a vast number of potentially hazardous mixtures using experimental animals is not a practical approach [30].

2.2. The Shift Towards Effect-Based Methods in Risk Assessment

Environmental risk assessment has evolved to account for the growing recognition of the complexity of interactions of pollutants, the ecological significance of sublethal contaminant effects, and the necessity of evaluating cumulative risks from multiple stressors. As a result, environmental risk assessment has increasingly shifted from purely chemical analysis to a stronger focus on biological effects [33]. In particular, the use of multiple biomarkers has gained attention for its ability to clarify toxicity and interactions of pollutants with other environmental factors. Biomarkers have primarily been developed and applied to assess water quality in surface water, wastewater, and recycled water, where biological activity is often detected at relatively high levels [34]. However, the implementation of effect-based methods is also highly effective for screening water samples with extremely low concentrations of individual contaminants. This effectiveness stems from the unique capability of biomarkers to measure the cumulative biological response resulting from all contaminants present in a sample. Establishing connections between biomarkers at molecular and biochemical levels and organismal responses is crucial for effective risk assessment. This approach integrates the sensitivity and specificity of molecular and biochemical indicators with ecologically relevant outcomes, enabling early detection of environmental damage, assessment of its severity, and the implementation of measures to safeguard ecosystem health [33].

2.3. Adverse Outcome Pathways (AOPs) Are Key Components in Risk Assessment

To enhance chemical risk assessment, the US Environmental Protection Agency has implemented models known as Adverse Outcome Pathways (AOPs) [35]. Furthermore, the European Chemicals Agency adopts AOPs to guide decision making in accordance with the REACH regulation [36]. AOPs outline how toxic effects develop across different biological levels by linking mechanistic biological responses to significant adverse impacts on entire organisms and populations. This methodology supports the integration of emerging scientific insights and assessment techniques such as in vitro assays and toxicological modelling, potentially minimising or replacing the need for whole-organism testing [37].

2.4. Advances in Risk Assessment with New Approach Methodologies (NAMs)

In addition to AOPs, New Approach Methodologies (NAMs) enhance and strengthen chemical risk assessment by utilising protective and relevant models [38]. This advancement also aligns with and incorporates the concept of phytotoxicology, thus optimising the use of other species and systems in place of higher evolved organisms through the translation of conserved mechanisms across taxa. Regulatory agencies across the globe acknowledge the significance of promptly adopting suitable NAMs for hazard and risk assessment, implementing adaptable, efficient, and scientifically robust processes to build confidence in utilising NAMs for regulatory decision-making [39]. In doing so, regulatory bodies are automatically responding to key animal welfare concerns and have the potential to drive significant scientific advancements and, in some cases, offer economic benefits [38]. This involves more relevant methodologies and tools that reflect species-specific biology. For instance, employing cells or tissues from aquatic species, such as Danio rerio, Daphnia magna, or Oryzias latipes, can help the modelling of key biological pathways and uncover crucial underlying and conserved mechanisms of action [23,25,26,27,38,40,41,42].

3. Water Fleas as Key Species in Risk Assessment

The cladoceran Daphnia has been widely recognized as a reliable bioindicator species in environmental risk assessment and ecotoxicology since the 1980s [43]. Found in lakes and ponds across all continents, daphnids play a crucial role in food-web dynamics, making it an essential organism for assessing ecosystem health and pollutant impacts. As a pelagic filter-feeding zooplankter with the potential for rapid population growth, daphnids serve as both a primary consumer of phytoplankton and a key food source for secondary consumers. This dual role establishes daphnids as a significant ecological interactor, with profound effects on the structure and functioning of aquatic ecosystems [44].

In the context of environmental risk assessment, daphnids are particularly valuable due to their influence on multiple ecological processes, including phytoplankton grazing, nutrient cycling, and their position as a preferred prey species for higher trophic levels. These processes are critical for evaluating the broader ecological implications of pollutants, particularly those that affect water quality, such as heavy metals, pesticides, and other contaminants. Daphnids’ sensitivity to changes in environmental conditions, coupled with its role in regulating nutrient cycling and maintaining the balance of aquatic food webs, makes it an ideal indicator for assessing the impacts of environmental stressors.

Furthermore, daphnid populations display a range of morphological, physiological, and behavioural adaptations that can alter the strength of these ecological interactions. These adaptations, which are known to exhibit genetic variability [45], further enhance daphnids’ utility in risk assessments, as they provide insight into the organism’s resilience and capacity to respond to environmental stress. Consequently, the use of daphnids as a bioindicator species offers valuable information for understanding the potential risks posed by contaminants, guiding management practices to protect aquatic ecosystems.

In chemical risk assessment, daphnids are commonly used as model organisms due to several key advantages: their small size, ease of culture in typical laboratory settings, body transparency, and rapid generation time. All of which facilitate rapid and cost-effective testing. As a result, daphnids are an ideal species for routine monitoring in water risk assessments. Their high sensitivity to environmental changes and stressors makes them a valuable tool in ecotoxicology and ecology research, where they are employed to evaluate the effects of pollutants on aquatic ecosystems. Beyond the well-established OECD protocols for acute toxicity [46] and reproduction [47] in chronic exposures, additional phenotypic markers such as survival, growth, feeding (ingestion) performance and behavioural changes (i.e., swimming), are commonly assessed to indicate responses to toxicants. Furthermore, molecular responses (i.e., gene expression, biochemical markers, and immune responses), are also frequently assessed to gain a comprehensive understanding of how contaminants affect daphnid populations.

These bioassays provide valuable insights into the impacts of a variety of contaminants prevalent in aquatic ecosystems. By utilizing daphnids as a model organism in water quality assessments, researchers contribute to the development of new regulatory guidelines and legislation aimed at improving water quality and environmental protection across numerous categories of pollutants. This approach not only enhances our understanding of aquatic toxicology but also supports the creation of effective policies for safeguarding aquatic ecosystems.

3.1. Molecular Perturbations in Response to Pharmaceuticals

With the overall increase in the life expectancy of the human population and along with the increased accessibility to pharmaceuticals and self medication, pharmaceuticals enter the aquatic environment from many routes. Whether from direct human activities such as the improper disposal of drugs or the use of pharmaceuticals in relation to animals (i.e., animal farming), active pharmaceutical ingredients and their metabolites find their way in the aquatic ecosystem. Pharmaceuticals are designed to be biologically active at low doses, making even trace levels environmentally significant. This, combined with incomplete removal in WWTPs, has resulted in their increasing detection in surface waters and effluents worldwide. Consequently, pharmaceuticals are now recognised as a rapidly emerging group of contaminants in freshwater systems. They have been found to exert significant biological effects on a plethora of non-target aquatic organisms, such as daphnids, often at sublethal or chronic exposure levels [48,49,50].

This has not only led to the detection of pharmaceuticals at concerning concentrations, but also to the emergence of a wide range of pharmaceutical metabolites, many of which remain unregulated and lack standardized methods for risk assessment—particularly at low, environmentally relevant doses.

3.1.1. Antibiotics

Antibiotics can enter the environment and act as environmental stressors to aquatic organisms with interactions in host-microbiota and the diversity of gut bacteria. Changes in the microbiome are observed when the organisms are exposed to pharmaceuticals. Antibiotics such as aztreonam and sulfamethoxazole can alter the microbiome taxa and impact fecundity negatively [51]. For example, tetracycline, which, similar to sulfamethoxazole, is a broad-spectrum antibiotic against both gram-positive and gram-negative bacteria, changes the bacterial abundance and impairs reproduction and survival in daphnids [52]. On the other hand, antibiotics may have stimulatory effects on the host through the suppression of abundant taxa, which allows the growth of other taxa. Low concentrations of ciprofloxacin increases the antioxidant capacity, growth, and fecundity, thus, suggesting the complexity of early effect symptoms of antibiotic exposure [53]. The exposure of daphnids to the variety of antibiotics may also contribute to the worldwide concern of antimicrobial resistance. When cultures are grown with bacterial communities containing the resistant gene tet(A), the gene takes refuge in the animal’s gut where they have the potential to further proliferate and horizontally transfer genes to the natural microbiota of the daphnids. Antimicrobial resistance is then exacerbated when the daphnids migrate to other freshwater areas [54]. Beyond daphnids, this phenomenon has also been observed in other terrestrial, marine and freshwater organisms. Other invertebrates are a source of antibacterial genes, but in particular, vertebrate organisms such as birds, fishes and amphibians give refuge to larger populations of bacteria in their tissues with antibiotic resistance. In fish, these genes are typically found in the gut and skin microbiota. In the safety of the organism and shielded from the external environment, the bacteria carrying the gene can reside for long durations, and can easily spread to other areas as the animal travels. This risk of organisms acting as reservoirs and vectors of antibiotic resistance across trophic levels mirrors those of daphnids [55,56,57].

3.1.2. Antiepileptics

Antiepileptic drugs have been used for the treatment of convulsive disorders. Typical representatives found in the environment include carbamazepine, gabapentin and valproic acid. Carbamazepine has shown dose-dependent effects hindering food ingestion, body length, growth, and reproduction by decreasing breeding frequency and the total number of eggs laid in daphnids. Daphnids also display abnormal phototactic behaviour upon exposure, increasing their chance of falling prey to predators in the environment. This also results in the alterations of enzyme activities such as acetylcholinesterase, superoxide dismutase, catalase and glutathione reductase, alkaline phosphatase, lactate dehydrogenase, lipase, glutathione-S-transferase, and β-galactosidase activity, causing neurotoxicity and oxidative stress for the daphnids which is exacerbated by bioconcentration [58,59,60]. The effects of other drugs such as gabapentin on biochemical markers have also been investigated on daphnids, where it was shown to impact acid phosphatase, peptidase, lactate dehydrogenase, lipase, and glutathione-S-transferase activity as well [60]. Valproic acid exposure also hindered reproduction and decreased the number and viability of offspring [61].

3.1.3. Antidepressants

With the ongoing increase observed in mental health disorders, antidepressants have been more widely introduced in recent years. Sertraline for example is a common antidepressant which can alter the expression of vitellogenin, disrupt moulting, increase the number of offspring, and cause reproductive and serotonergic dysfunction. This effect can persist for several generations even when cultures are no longer exposed to the stressor. In fact, the removal of sertraline also caused the overcompensation of sert expression in the recovery generation [62]. On the other hand, in subsequent generations, sertraline can then decrease the number of offspring. Venlafaxine was assessed in the same chronic experiment, again resulting in an effect on reproduction, but decreasing offspring numbers in the first generation before the daphnids build up a tolerance [63]. Increase in reproduction is also shown in daphnids exposed to fluoxetine due to the endocrine disruption. The effects of antidepressants are also heightened by being combined with other stressors such as low food conditions [64]. However, other literature also reports a decrease of offspring after exposure, reiterating the importance of dosage [65].

3.1.4. Antidiabetics

Diabetes is a major disease which affects a significant number of people on a global scale. Many drugs are used to regulate blood sugar and metformin is the usual treatment prescribed for type 2 diabetes. In daphnids, metformin and its metabolite guanylurea exhibit low toxicities relative to their environmental concentrations [66]. Literature has even reported that metformin can activate signalling pathways involved in gene expression and protein synthesis and increase the hypoxic tolerance of the animal, and this can be advantageous during environmental stress [67]. However, despite this positive effect, it has a dose dependent effect and can cause toxicity, reproduction, affect ingestion rate, and shorten the lifespan of exposed daphnids at higher concentrations [68,69,70]. Biochemical markers like an alteration in enzyme activity such as alkaline phosphatase, glutathione-S-transferase, and β-galactosidase also reiterate the negative impact of metformin exposure [71].

3.1.5. Non-Steroidal Anti-Inflammatory Drugs (NSAIDs)

NSAIDs are extensively utilized pharmaceuticals worldwide which emerged as significant contaminants in water, soils, and sewage sediments as a result of their improper discharge and disposal. Much progress has been achieved in their study in the environment [72]. Typical examples include indomethacin and ibuprofen, which have been assessed on daphnids and reveal a decrease in feeding performance upon exposure to their mixture, as well as changes in key enzyme activities to both individual and mixture exposures. Alkaline and acid phosphatases, lipase, peptidase, β-galactosidase, and glutathione-S-transferase activity was impacted, and the effect was further enhanced in transgenerational exposures until the stressor was removed from the daphnids [73]. In acute experiments, naproxen is also another NSAID that has shown to negatively impact daphnids. It can exert toxicity [74], and antioxidant enzymatic activities which served as biochemical markers were significantly different to the control. Signalling pathways and the antioxidant system were affected, and development, reproduction, and behaviour were hindered [75]. The most commonly prescribed NSAID which comes in many forms from tablets to ointments is diclofenac. Diclofenac has been detected in significant levels in the aquatic environment and it has been studied extensively for its adverse effects. It was included in the European priority substances watchlist in 2015 and it affects many non-target species. The pharmaceutical has a time-dependent and concentration-dependent toxicity on daphnids. Sublethal effects such as a change in body length, decrease in egg production, and significant reduction in the number of carapaces per adult water flea were also observed [76]. The negative impact of the NSAID is similarly supported in other studies which recorded a decrease in growth and reproduction, but also a delay in getting the first brood and egg production, inhibition in swimming activities, heart rate, and thoracic limb activities. Diclofenac also introduced metabolic perturbations which include the decrease in citrate and aconitate from the TCA cycle [71]. Additionally, genes related to metabolism, growth, development, reproduction and antioxidant response were inhibited. Specifically, the alteration of the reproductive parameters exhibited in the transgenerational experiments suggest that diclofenac is possibly an endocrine disruptor [77,78].

3.1.6. Beta-Blockers

Propranolol is a common example of a beta-blocker that has shown to be toxic to daphnids at high concentrations [74]. Specifically, it was reported that there was increased lipid production which resulted in an increase in the number of offspring when compared to the control. This enhanced reproduction was linked with promoting certain signalling compounds [79]. Metoprolol has been also assessed for its impacts on daphnids, and it was found that high doses resulted in decreased heart rate which can reduce fecundity and size at first neonate production when continuously exposed to multiple generations [80]. Pharmaceutical mixtures containing bisoprolol and sotalol also induced immobilisation in a small proportion of daphnids [81].

3.2. Molecular Perturbations in Response to Nanomaterials

With the rapid innovation in the field of nanotechnology, nanomaterials have been dramatically increasing in their applications in the last decade. From commonly consumed products to food packaging and other sectors, nanomaterials increasingly lead to continuous exposure to humans and the ecosystem. Similarly to other emerging contaminants such as pharmaceuticals, despite their growing prevalence, current methodologies specific to nanoparticles’ detection are limited, their mechanisms of toxicity are still not completely understood, and their ecological impacts remain underexplored [82]. The main type of nanomaterials explored in the context of ecotoxicology revolve around elements such as carbon and metals, all which come in different forms such as particles or in different shapes and surfaces. These materials possess unique physicochemical characteristics, such as a high surface area, reactivity, and the ability to form aggregates that influence their bioavailability, environmental fate, and interactions with the aquatic ecosystem and its inhabitants [83,84].

3.2.1. Carbon Nanomaterials

Carbon dots are carbon-based nano materials and are considered relatively environmentally friendly [85]. However, research has shown that it caused developmental toxicity and oxidative stress, plus it impacted the feeding behaviour, digestive system, and symbiotic bacteria in water fleas. Specifically, their ingestion caused an imbalance of normal host-microbiota interactions through the reduction of microorganisms involved in energy metabolism and altered how the immune system of daphnids interacted with its microbiome [86]. Additionally, pneumatosis cystoides intestinalis, which is the accumulation of gas in cysts within the intestinal walls, was also observed in daphnids [87]. Fullerenol nanoparticles, which have many biomedical applications, are another carbon-based nanomaterial that can accumulate in the intestines and head of daphnids and cause toxicity [88]. Upon co-exposure with UV light, antioxidant enzyme activities were increased. In another study, β-galactosidase, trypsin, cellulose, and amylase, enzymes that aided in digestion were decreased. They are also capable of causing oxidative stress, hinder the moulting process, decrease digestion rate, filtration rate, and the number of neonates produced. The reduction in fitness can potentially damage population dynamics and lead to bioaccumulation [89,90].

3.2.2. Metal Oxide-Based Nanoparticles (MNPs)

MNPs are gaining widespread traction due to their range of applications; however, they are getting introduced into our freshwater systems and adding to the global pollution concern. Aluminium oxide nanoparticles are one common MNP, and they are inorganic nanomaterials that can be toxic to daphnids as reported by ecological risk assessments. Certain crystalline phases were shown to be more toxic and were able to accumulate in the intestines of the animals. Cellular uptake of the nanoparticles caused impacted body length, age at first brood, number of neonates per surviving adult, and the production of reactive oxygen species, and in comparison to control groups, had swimming behaviour when under chronic toxicity tests [91]. Zinc oxide is another MNP that has been evaluated for its toxicity in daphnids. Investigations have shown that it similarly has the ability to accumulate in the gut and displays high toxicity, with a 50-ppm concentration for 48 h as its lethal dose [92]. The accumulation of the MNP is due to the difficulty of depuration, as suggested by acute experiments [93]. The inhibition of anti-predator defences in daphnids were another negative impact from exposure to MNPs. Silver nanoparticles caused a reduction in anti-predator defence mechanisms when exposed to predator kairomones in daphnid neonates [94]. Studies also reveal the reduction of reproduction, growth, and survival rates as well as aging of the heart, abdomen, and abdominal claw due to antioxidant stress from the exposure. Disruption in moulting and deformities such as impaired eye and tail development were visible in exposed cultures, and severe cases where daphnids completely lack a tail were also observed. These effects persisted across multiple generations, suggesting disruptions in the embryonic development from the silver nanoparticles. These effects are explained at the gene-level analyses, where it was revealed silver nanoparticles were capable of upregulating and downregulating a variety of genes [95].

4. Translation of Molecular Findings to New Metrics for the Early Prediction of Pollution

Daphnids have proven to be invaluable bioindicators to assess the impact of pollutants on non-target organisms, and by extension, the protection of aquatic environments. Their physiological, behavioural, and molecular responses offer crucial insights into water quality and pollutant toxicity (Figure 1). Extensive studies highlight their sensitivity to contaminants, underscoring their potential role in sensitive water quality monitoring and pollution prevention. Moreover, the effects of pharmaceuticals and nanomaterials observed in daphnids are not limited to these organisms alone, but they reflect the broader implications for aquatic ecosystems. Additionally, the detection of pollutants in daphnids suggests their presence in the environment, with contaminants likely present in producer organisms, such as algae, which daphnids consume. Given their position in the lower trophic levels of the aquatic food web, daphnids facilitate the bioaccumulation of harmful compounds in higher organisms, including fish and potentially humans.

Increasing our understanding over the responses of daphnids to pollution and stressors provides essential insights into the long-term ecological consequences of pharmaceutical and nanomaterial pollution, such as shifts in population dynamics and trophic interactions. Monitoring daphnids helps to establish new metrics for evaluating the effects of environmental contamination on aquatic ecosystems. Integrating daphnid-based models with traditional monitoring systems holds promise for developing early warning systems to detect emerging contaminants and mitigate widespread ecological damage. The ongoing optimization of daphnid models, coupled with advancements in assay miniaturization, makes these approaches not only more efficient but also more accessible, economical, and capable of supporting repeated testing across a wide array of contaminants (Figure 1). These refinements in bioassays further reinforce the critical role of daphnids in detecting pharmaceuticals, nanomaterials, and other pollutants [96]. Future research should focus on expanding the use of daphnid-based bioassays for a broader range of pollutants, particularly emerging contaminants. Additionally, efforts to further enhance the sensitivity and cost-effectiveness of these models will be crucial in advancing early detection and preventing ecological damage, and elevate daphnids as “an equivalent to a canary in the coal mine for the early prediction of pollution”.