The Silent Threat: Exploring the Ecological and Ecotoxicological Impacts of Chlorinated Aniline Derivatives and the Metabolites on the Aquatic Ecosystem

The growing concern over the environmental impacts of industrial chemicals on aquatic ecosystems has prompted increased attention and regulation. Aromatic amines have drawn scrutiny due to their potential to disturb aquatic ecosystems. 4-chloroaniline and 3,4-dichloroaniline are chlorinated derivatives of aniline used as intermediates in the synthesis of pharmaceuticals, dyes, pesticides, cosmetics, and laboratory chemicals. While industrial applications are crucial, these compounds represent significant risks to aquatic environments. This article aims to shed light on aromatic amines’ ecological and ecotoxicological impacts on aquatic ecosystems, given as examples 4-chloroaniline and 3,4-dichloroaniline, highlighting the need for stringent regulation and management to safeguard water resources. Moreover, these compounds are not included in the current Watch List of the Water Framework Directive, though there is already some information about aquatic ecotoxicity, which raises some concerns. This paper primarily focuses on the inherent environmental problem related to the proliferation and persistence of aromatic amines, particularly 4-chloroaniline and 3,4-dichloroaniline, in aquatic ecosystems. Although significant research underscores the hazardous effects of these compounds, the urgency of addressing this issue appears to be underestimated. As such, we underscore the necessity of advancing detection and mitigation efforts and implementing improved regulatory measures to safeguard the water bodies against these potential threats.

Chloroanilines, such as 4-chloroaniline (4-CA) and 3,4-dichloroaniline (3,4-DCA), are used as building blocks in the synthesis of pharmaceutical products such as chlorhexidine and triclocarban, used in the production of antiseptic mouthwashes, deodorants, soaps, etc. [6,7].These aromatic amines play a crucial role in the development of active pharmaceutical ingredients and are incorporated into the chemical structures of diverse drug classes, including analgesics, antipyretics, and antivirals [8].The vibrant coloration in many dyes and pigments is also attributed to the presence of aromatic compounds such as chloroanilines [9].These aromatic amines are used as precursors in the production of azo dyes, which are widely used in the textile, printing, and cosmetic industries [10].They are also utilized in the synthesis of agrochemicals, such as herbicides and fungicides, which play a vital role in crop protection and enhancement [11].The significance of chloroanilines in these industrial sectors underscores their economic importance and the extensive use of these aromatic amines in various manufacturing processes [4,5].However, it is essential to consider the potential environmental impacts associated with their production, use, and disposal [12].
The chemical processes involved in the synthesis of 4-CA and 3,4-DCA certainly have associated challenges and potential environmental problems.Chemical waste can include spent catalysts, unreacted starting materials, and byproducts that are not part of the desired final product, degrade slowly, bioaccumulate, contaminate the water, and impact soil health [12].High concentrations of compounds used in the synthesis of 4-CA and 3,4-DCA can harm ecosystems through air pollution and soil damage, for example, via eutrophication, when excess nutrients are introduced in the soil, which can disrupt the balance of nutrients, negatively impacting the growth and health of plants, or altering the structure and functioning of soil microbial communities [20,21].This can have cascading effects and consequently lead to biodiversity loss [22].Herbicides based on 4-CA and 3,4-DCA can leach into groundwater and induce poor health conditions in the individuals exposed.Some of these compounds disrupt hormone production and are potentially genotoxic, mutagenic, and carcinogenic [23,24].On the other hand, the acidification of water bodies caused by the substances involved in the synthesis of 4-CA and 3,4-DCA is also a concern.As the pH of water decreases, aromatic amines properties and bioavailability can be affected through their protonation, leading to a decrease in their solubility and potentially altering the speciation and transformation of these compounds, which can impact their toxicity and persistence in the environment [25].
These risks necessitate careful waste management practices to prevent contamination of water and soil and the release of harmful substances into the atmosphere.Moreover, workers' exposure to these chemicals must be carefully managed to prevent health risks.However, the specific risks and waste products depend on the methods used and cannot be universally defined, depending on various factors, including the starting materials, intended applications, specific methods, and conditions used in the synthesis process [26][27][28][29].Due to the lack of information concerning the environmental presence and impact of aromatic amines (compounds with high and diverse use) on aquatic ecosystems, this opinion paper intends to review the information available concerning these two compounds (4-CA and 3,4-DCA), identifying their environmental effects on aquatic organisms and measures to be taken into consideration to safeguard the environment.

Environmental Presence
The entrance of 4-CA and 3,4-DCA into the environment can occur through various pathways, including wastewater discharges, accidental spills, and atmospheric deposition [30].These pathways can lead to the contamination of water bodies and pose risks to aquatic organisms.Even after undergoing treatment, trace amounts of these chemicals can still be present in wastewater-discharged effluents, potentially affecting downstream ecosystems [31].In Table 1, environmentally relevant concentrations (ERC) of 4-CA and 3,4-DCA, already detected worldwide and from different aquatic matrices, are evidenced [32][33][34][35][36][37][38][39][40].The information resumed in Table 1 is a result of a literature review  where specific keywords were used: aromatic amine, chloroaniline, 4-chloroaniline, 4-CA, p-chloroaniline, 3,4-dichloroaniline, 3,4-DCA, drinking water, wastewater influent, wastewater effluent, river, superficial water "https://scholar.google.com/(accessed on 15 June 2023)".water bodies caused by the substances involved in the synthesis of 4-CA and 3,4-DCA is also a concern.As the pH of water decreases, aromatic amines properties and bioavailability can be affected through their protonation, leading to a decrease in their solubility and potentially altering the speciation and transformation of these compounds, which can impact their toxicity and persistence in the environment [25].These risks necessitate careful waste management practices to prevent contamination of water and soil and the release of harmful substances into the atmosphere.Moreover, workers' exposure to these chemicals must be carefully managed to prevent health risks.However, the specific risks and waste products depend on the methods used and cannot be universally defined, depending on various factors, including the starting materials, intended applications, specific methods, and conditions used in the synthesis process [26][27][28][29].Due to the lack of information concerning the environmental presence and impact of aromatic amines (compounds with high and diverse use) on aquatic ecosystems, this opinion paper intends to review the information available concerning these two compounds (4-CA and 3,4-DCA), identifying their environmental effects on aquatic organisms and measures to be taken into consideration to safeguard the environment.

Compound
Type River-Superficial water Zonguldak, Turkey 0.00066-0.00082[35] 3,4-DCA water bodies caused by the substances involved in the synthesis of 4-CA and 3,4-DCA is also a concern.As the pH of water decreases, aromatic amines properties and bioavailability can be affected through their protonation, leading to a decrease in their solubility and potentially altering the speciation and transformation of these compounds, which can impact their toxicity and persistence in the environment [25].These risks necessitate careful waste management practices to prevent contamination of water and soil and the release of harmful substances into the atmosphere.Moreover, workers' exposure to these chemicals must be carefully managed to prevent health risks.However, the specific risks and waste products depend on the methods used and cannot be universally defined, depending on various factors, including the starting materials, intended applications, specific methods, and conditions used in the synthesis process [26][27][28][29].Due to the lack of information concerning the environmental presence and impact of aromatic amines (compounds with high and diverse use) on aquatic ecosystems, this opinion paper intends to review the information available concerning these two compounds (4-CA and 3,4-DCA), identifying their environmental effects on aquatic organisms and measures to be taken into consideration to safeguard the environment.

Environmental Presence
The entrance of 4-CA and 3,4-DCA into the environment can occur through various pathways, including wastewater discharges, accidental spills, and atmospheric deposition [30].These pathways can lead to the contamination of water bodies and pose risks to aquatic organisms.Even after undergoing treatment, trace amounts of these chemicals can still be present in wastewater-discharged effluents, potentially affecting downstream ecosystems [31].In Table 1, environmentally relevant concentrations (ERC) of 4-CA and 3,4-DCA, already detected worldwide and from different aquatic matrices, are evidenced [32][33][34][35][36][37][38][39][40].The information resumed in Table 1 is a result of a literature review  where specific keywords were used: aromatic amine, chloroaniline, 4-chloroaniline, 4-CA, p-chloroaniline, 3,4-dichloroaniline, 3,4-DCA, drinking water, wastewater influent, wastewater effluent, river, superficial water "h ps://scholar.google.com/(accessed on 15 June 2023".≤20.19 Tres Rios Wetlands, Gila River, USA 0.17 [36] Homestead, Florida, USA 0.17 Sado River basin, Portugal ≤0.71 [40] In addition, accidental spills during the transportation, storage, or handling of chloroanilines can lead to direct contamination of water bodies and, consequently, to immediate exposure in nearby aquatic ecosystems, with potential toxic effects on organisms [41].These compounds can also be released into the atmosphere during industrial operations, combustion processes, or volatilization from contaminated surfaces.Once in the air, chloroanilines can be transported over long distances and eventually be deposited into land or water surfaces through precipitation or dry deposition, contaminating remote water bodies, even in areas far away from the pollution source [42].4-CA and 3,4-DCA can undergo diverse transformation processes (e.g., reductive dichlorination and microbial mineralization), may exhibit relative stability, adsorb to suspended particles in water bodies, and have leaching potential [43,44].
These compounds are considered persistent in the environment regarding the chemical properties in water.4-CA presents a boiling point of 232 • C, a vapor pressure of 0.015 mm Hg at 25 • C [11], it can be volatilized (35.7 days half-life in rivers), photo oxidized in surface water (1 to 3 h half-life with low organic matter) and is biodegraded (several days to months half-life) [45].3,4-DCA showed a boiling point of 272 • C, vapor pressure of 1 mm Hg at 81 • C [46], and no evidence of hydrolysis, volatilization, and biodegradation.However, it undertakes photolysis in surface water (18-day half-life) and can bioaccumulate in groundwater sediments due to its very slow degradation rate (1000 days half-life) [47].

Impact on Aquatic Organisms
The freshwater biodiversity threat refers to the substantial decline in species diversity and abundance within freshwater ecosystems, including rivers, lakes, and wetlands [48].This occurs due to multiple factors, including habitat loss, water pollution, invasive species, overfishing, climate change, altered water flow, and insufficient conservation efforts [49].The repercussions of this crisis extend widely, affecting ecosystem services and human livelihoods alike [50].Successfully addressing the environmental impacts is important for a comprehensive approach involving habitat protection, pollution management, sustainable water practices, invasive species control, and international cooperation [51].
Aromatic amines, despite being utilized in industrial and commercial sectors, present a notable hazard to freshwater ecosystems and can exacerbate the ongoing biodiversity threat.Entering water bodies through diverse pathways, these compounds endure, resulting in water pollution [52].Exhibiting toxicity to aquatic organisms induces species depletion, degrades habitats, and fosters bioaccumulation [12].Regarding these effects, it is urgent to implement measures to mitigate the impact of aromatic amines on the preservation of freshwater biodiversity.
Table 2. Literature review of ecotoxicological effects in different species after exposure to 4chloroaniline (4-CA), 3,4-dichloroaniline (3,4-DCA), linuron and diuron (parent compounds and/or byproducts of 4-CA and 3,4-DCA).Considering this literature review (Table 2), behavioral changes (e.g., feeding patterns, locomotion, predator-prey interactions, and avoidance), as well as the disruption of growth, reproduction, and development of aquatic organisms (e.g., reduced growth rates, gamete production, fertilization, and impaired development) after exposure to xenobiotics, can impact the ability of individuals to find food, evade predators, reduce fitness and reproductive capacity [64].This can ultimately affect survival and reproductive success, having long-term implications for aquatic ecosystems' population dynamics and health [70].However, the literature has limited information on the ecotoxicological impacts of 4-CA and 3,4-DCA at environmentally relevant concentrations.

Status in Water Framework Directive
The Water Framework Directive (WFD, adopted in 2000) is a legislation in the EU focused on water policy that aims to achieve and maintain all water bodies with a good ecological and chemical status.Both 4-CA and 3,4-DCA compounds are listed as candidates for the 4th Watch List under the WFD [71].4-CA and 3,4-DCA were originally selected as Priority 1, but after Member States and Stakeholder experts' input regarding uncertainties considering the predicted no-effect concentration (PNEC), Joint Research Center experts chose to gather more data before including them in the Watch List, being moved into the Priority 2 category [71].Priority 1 and Priority 2 substances contain the "most suitable candidates for inclusion in the next Watch List" and "almost suitable candidates for the next Watch List, but for which a reliable PNEC or analytical method is missing".However, these two categories include compounds that may represent a risk in the aquatic compartment, having limited or low-quality monitoring data in the EU for risk assessment.

Environmental Concerns about Aromatic Amines
Detecting and measuring chemicals such as aromatic amines in the environment poses several challenges due to the low concentrations (ng/L to µg/L), complex matrices, and the need for sensitive and selective analytical quantification techniques.However, advancements in analytical methods, through the implementation of specific techniques such as a derivatization step prior to gas chromatography (GC), extraction processes (e.g., liquid-liquid extraction or solid-phase extraction) followed by high-performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE) and spectrophotometric methods, can provide improved capabilities for the detection of these specific compounds in natural waters [72].
Moreover, it is crucial to address the issue of contaminated water sources and implement appropriate measures to reduce the exposure of aquatic individuals to aromatic amines.The specific mechanism of action of aromatic amines on aquatic environments and organisms cannot be understated.The evidence gathered from scientific studies, such as the potential for long-range transport and the persistence of aromatic amines in the environment, highlights the need for stringent regulation and management practices to prevent the release of these chemicals into water bodies [73].Efforts should focus on reducing the use with the promotion of alternative substances, for instance, non-halogenated anilines (e.g., methylanilines, nitroanilines, or methoxyanilines) or less toxic aromatic compounds, as intermediates in the industry where chloroanilines are currently used [74].On the other hand, it is important to implement effective wastewater treatment processes that have been referred to in the literature for aniline removal, such as Electro-Fenton ® and peroxycoagulation processes using a flow reactor, implementing the use of a sequencing batch reactor, zero-valent ion coupled with hydrogen peroxide, among others [75][76][77].These methods can reduce the concentration of aromatic amines in wastewater treatment plant (WWTP) effluents, mitigating the impact on the ecosystems.Striving for a sustainable future demands proactive measures to protect water resources and the delicate ecosystems they support.

Future Developments
The big challenge for the future is to reduce the impacts of anthropogenic activities on natural resources and protect ecosystems to achieve an ecologically sustainable environment.Given the wide use of these compounds in industry for different purposes, as well as the wide dissemination to the environment via elimination processes or as a result of degradation products, it adds greater concern to the environmental presence.Additionally, it is empirical to anticipate the environmental impacts of aromatic amines before their undue occurrence, employing more effective analytical methodologies to study the interrelation between the health of organisms, even at sub-individual levels (more sensitive and early warning responses) and the ecosystem.A response at sub-individual levels to stressors, i.e., at the cellular level, such as perturbations of oxidative metabolism, after exposure to these compounds, can potentially initiate outcomes at individual levels, manifesting as changes in behavior and reproductive irregularities or even death.In such scenarios, a cascade effect can incite implications at the population level, such as altered abundance, species decay, or population dynamics.Consequently, sequential effects on the community level may be detected, such as disruption in food chains, alterations in community composition, or changes in biodiversity.Lastly, these influences can scale up to the ecosystem level, inducing functional changes such as nutrient cycling disturbance, a shift in energy flow, or alteration in ecosystem services [78].Therefore, effective anticipatory measures require complex and comprehensive prospective risk assessments, taking into consideration this complex web of interactions.
Although in the Water Framework Directive, 4-CA and 3,4-DCA are still not included as compounds for monitorization to ensure a good ecological water status, they are currently detected between the ng/L and µg/L range, as evidenced in Table 1.These environmentally relevant concentrations are also the threshold values to be taken into consideration for risk assessment and policies implemented for these compounds.It is also empirical to consider that at these concentrations, and as evidenced above, several harmful ecotoxicological effects are reported at the sub-individual and individual level in several organisms and populations, including in the aquatic compartment.Therefore, these findings cannot be disregarded.
Considering that WWTP can only partially remove these compounds from wastewater [31] and the production and consumption of aromatic amines has increased in recent years [5], this will likely lead to an increase in the concentrations in surface waters.In this case, an environmental risk assessment concerning aromatic amines becomes crucial for the implementation of policies to restrict their usage and diffusion through the aquatic environment.Particularly for compounds with low rates of degradation and directly applied in the environment, that can have a more detrimental impact on aquatic organisms.
Due to its wide use, the diverse possibilities of reaching the environment, persistence, analytical methods of analysis (often inappropriate), and the lack of scientific evidence on the real risks to aquatic ecosystems (considering the above factors) reinforce the silent threat that aromatic amines, mainly 4-CA and 3,4-DCA, represent for a sustainable environment balanced with anthropogenic needs.