**3.3 Toxicokinetics**

The prevalence of organic organophosphates in living organisms has become a research target. Toxicological research has suggested that chronic exposure may be directly related to damage to the endocrine system, with a high risk of reproduction being impaired. These compounds may also be related to systemic toxicity, affecting other important organs [101].

Unintentional dust ingestion and skin absorption are the routes with the highest organic organophosphate absorption rate. How these compounds are distributed in biological tissues has not been completely elucidated. However, the highest organophosphates levels have been found in the liver, muscle, and gonads [100].

Compounds are biotransformed through metabolization to diesters and hydroxylation by phase I reactions, followed by conjugation with glucuronide and sulfate in

phase II reactions. These reactions happen both in humans and other living beings. Biotransformation products are important for monitoring and evaluating exposure to organophosphates: these products are eliminated fast because they are very hydrophilic [105].

Although organophosphate metabolism is relatively fast, even low concentrations of the metabolites may have physiological and endocrine effects. Considering that human exposure to these compounds is high and that multiple exposure pathways exist, the concern is enormous. For instance, children are generally less able to metabolize and to excrete xenobiotics, so these compounds are more toxic to them [106].

#### **3.4 Toxicological effects**

Among the potential adverse effects of organic organophosphates, reproductive and neurological effects are highlighted. Although the toxicity mechanisms of these compounds have not been fully elucidated, the consequences of being exposed to them involve cell apoptosis, ROS production, membrane disturbance, and mitochondrial alterations, among others [100].

Neurotoxic effects are concentration-dependent and inhibit DNA synthesis, decreasing the number of cells and altering neural differentiation. Acetylcholinesterase (AChE) inhibition is another reported mechanism. AChE is a widely used marker in neurotoxicity studies. Tris-(1,3-dichloro-2-propyl) phosphate (TDCPP) and TPhP have an affinity for the nervous system and are commonly associated with neurotrophic factor inhibition [107].

Aquatic organisms are often exposed to organophosphates, which tend to be neurotoxic to them. Sun et al. [108] analyzed the neurotoxicity of the halogenated tris-(2 chloroethyl) phosphate (TCEP) and the non-halogenated alkyl tri-*n*-butyl phosphate (TNBP) in zebrafish (*Dario rerio*). More specifically, these authors analyzed locomotor behavior, enzymatic activity, and AChE gene transcription. They found that zebrafish exposure to these compounds in early life stages affects locomotor behavior and gene transcription, suggesting that exposure to organophosphates may be relevant in humans, especially in children.

Exposure at developmental stages may also affect cardiac development. Cardiac development comprises several stages for increasing formation during embryogenesis. When the process occurs properly, chamber formation and maturation, septation, and valve formation take place correctly [109].

Using zebrafish as a model species for studies on developmental toxicity is advantageous and has been gaining ground in several areas of toxicology. Alzualde et al. [110] used zebrafish as a model in developmental toxicity assays to test not only the cardiotoxicity but also the neuro- and hepatotoxicity of organophosphates. These authors found that TPhP affects the heartbeat and reduces locomotor activity and hepatic edema. These data are extremely relevant when it comes to human biomonitoring.

Abe et al. [111] evaluated the toxicity of halogen-free flame retardants in zebrafish to trace a toxicity profile. At all concentrations used, ALPi did not show any sublethal or teratogenic effects, suggesting that ALPi may be a good alternative for brominated flame retardants. However, further studies are still needed to support this information [111].

The crustacean *Daphnia magna* is also a widely used model for toxicity testing. To test the chronic toxicity of ALPi, Waaijers et al. [112] exposed *D. magna* by 21 for toxicity tests. The toxicity of ALPi increased with the time of exposure, with low acute toxicity and moderate chronic toxicity [112].

TDCPP and TPhP are also potential endocrine disruptors, altering hormone levels and decreasing semen quality in adult men. Furthermore, the concentration of these compounds in house dust has been correlated with decreased sperm concentration, increased prolactin level, and decreased free thyroxin (T4) level [113].

Given that reproductive system integrity also depends on the organism's redox state, An et al. [114] tested TPhP and TCPP cytotoxicity in HepG2, A549, and Caco-2 cells. In addition to inhibiting cell viability, these compounds increase ROS production, inducing DNA damage and mitochondrial dysfunction. These changes in redox balance may harm steroidogenesis and even estrogen metabolism, being directly related to reproductive changes.

Epidemiological studies on exposure to organophosphates are also gaining ground, especially when it comes to the early stages of development, when organophosphates may have greater consequences [115].

In a cohort study, Castorina et al. [116] evaluated how exposure to organophosphates, mainly TPhP, affected the cognitive or behavioral development of 310 schoolage children. The authors monitored exposure by analyzing metabolites present in pregnant women's urine. They observed decreased intelligence quotient and working memory, associated with an increased level of the urinary metabolite diphenyl phosphate (DPhP).

Although some compounds have been widely used in research, and even though much information is available, the effects of other types of organophosphates remain to be elucidated. For example, toxicity data on BPA-DP and RDP are limited, so their consequences on human health are unknown [82].

Monitoring organic organophosphate metabolites is necessary to assess and control biological exposure, not to mention that these metabolites may play important biological roles in the toxicity of these compounds. In any case, many studies on organic organophosphates are still needed to understand their toxic effects and to reduce exposure to them [117].
