Invertebrates' endocrine systems regulate many of the same processes as those of vertebrates, including growth, reproduction and development (Oehlmann and Schulte-Oehlmann 2003). The endocrine systems of invertebrates, including development and reproduction, have been discovered to be disrupted by several chemicals or combinations of compounds. The effects of endocrine disruptors on invertebrates may aid in predicting the potential endocrine-disrupting compounds responses in vertebrates (DeFur 2004). Because of their sensitivity to toxic chemicals, ease of manipulation, ease of culture, and short generation times, invertebrates have been excellent models for studying endocrine systems and toxicity testing. The current study was designed to evaluate the ability of atrazine to disrupt the endocrine system of P. clarkii. In order to determine the potential use of P.clarkii as a bioindicator for endocrine disruptor substance screening.
The results of the current study revealed a significant increase in the mortality rate of P. clarkii treated with lethal concentrations of atrazine. By increasing the concentrations and exposure times in both males and females, the mortality rate increased. The LC50 of atrazine for P. clarkii after 96 hours was determined to be 10.62 and 12.66 mg/l for adult male and female, respectively. It could be also noted that females are more tolerant than males when treated with atrazine. The death of P.clarkii may be due to the immunosuppressive effects of atrazine and the direct toxic effect of atrazine on immune cells (Galbiati et al. 2021). Galoppo et al. (2020) indicated that immunotoxic effects of atrazine affected by differences in gender and time, males more sensitive than females. These findings are consistent with the findings of Stara et al. (2018), who demonstrated that atrazine increased the mortality rate of the crayfish Cherax destructor and that LC50 value for atrazine was 12.1 mg/l after 96 hr. Omran and Salama (2013) demonstrated that the use of atrazine herbicides increased the mortality rate of Biomphalaria alexandrina.
The lowest concentrations chosen (LC10 and LC25) indicate a potential environmental concentration in the herbicide's upper environmental range, i.e., water contamination around treated feilds up to 1 mg/l (Graymore et al. 2001). Although the assayed atrazine concentrations are worst-case scenarios, determining their long-term impacts on reproductive parameters is important not only for the protection of P. clarkii, but also for future research aimed at establishing biomarkers in other decapod crustacean species and increasing our understanding of how this herbicide affects crustacean reproduction.
Estradiol (E2) is an estrogen steroid hormone and has an important role in the development and maintenance of female reproductive tissues. The present study showed that sublethal concentrations of atrazine increased level of Estradiol in ovary and haemolymph of P.clarkii. Similarly, Silveyra et al. (2018) found that Estradiol level increased in haemolymph of P. clarkii after exposure to Atrazine for one month. In this context, Mac Loughlin et al. (2016) who found that the Estradiol level increased in crayfish Cherax quadricarinatus after treatment with 2.5 mg/l atrazine. Although atrazine has been shown to raise estrogen levels by inducing aromatase activity in various vertebrate species (Hayes et al. 2006), no evidence of aromatase expression in crustaceans was already found (Swevers et al. 1991). Nonetheless, the herbicide under study may stimulate several other enzymatic pathways involved in oestrogen synthesis.
Progesterone is a steroid sex hormone that has an important role in embryogenesis. Atrazine increased haemolymph and ovary progesterone. Foradori et al. (2017) reported that atrazine affect progesterone level by centrally activating hypothalamic-pituitary-adrenal axis levels through the corticotropin-releasing hormone receptor.
Testosterone is an androgen that is found in the testis and is responsible for spermatogenesis. Males are sterile in the absence of testosterone or functional androgen receptors because spermatogenesis rarely progresses beyond meiosis.
(De Gendt et al. 2004). The present study showed that atrazine decreased testis and haemolymph testosterone level. This is in line with the findings of Silveyra et al. (2018) who reported that a decrease in testosterone level in haemolymph of Procambarus clarkii after exposure to atrazine. Omran and Salama (2013) found that the testosterone level decreased in hermaphrodite glans of B. alexandrina snail after treatment with sublethal concentrations of Atrazine. Also, these results were in harmony with Clair et al. (2012) who stated that atrazine decreased rat testosterone concentrations and proposed that atrazine is a potent endocrine disrupter that disrupts rat reproductive hormones. The decrement in testosterone levels may be due to the inhibitory effects of atrazine on the adrenal androgens synthesis (Zimmerman et al. 2014).
Proteins are crucial biochemical components that are required for metabolic pathways and biochemical reactions. Under extreme stress, protein provides energy in metabolic processes and biochemical activities. As a result, measuring total protein levels in the hemolymph, ovary, and testis could be used as a diagnostic tool to determine an organism's physiological state (Prasath and Arivoli 2008). The current study clearly demonstrated that total protein content was significantly reduced in the hemolymph, ovary, and testis of male and female P. clarkii after Atrazine treatment. Total protein levels in crayfish hemolymph and ovary and testis tissues may be depleted as a result of increased proteolytic activity in these organs or energy diversification to meet the impending energy demands during toxic stress. Furthermore, protein depletion could be linked to cell death or necrosis, which would result in a breakdown in the protein production machinery (Bradbury et al. 1987). Several studies have shown that atrazine has an inhibitory effect on total protein. Khan et al. (2016) reported a decrease of total protein in atrazine exposed freshwater fish Grass Carp Ctenopharyngodon idella. Akhtar et al. (2021) reported a decrease of total protein in Atrazine exposed snow trout Schizothorax plagiostomus. Opute and Oboh (2021) noticed a decrease in total protein in Clarias gariepinus following chronic Atrazine exposure.
Cytochrome P450 (CYP 450) is a critical biochemical marker and indication of some chemicals contamination (Jung et al. 2001) and has role in xenobiotic detoxification (Uno et al. 2012). The P450 family of arthropods encodes a wide range of enzymes involved in foreign chemical metabolism as well as endocrine and ecophysiological functions (Dermanw et al. 2020). The current study showed clearly that ovarian, testicular and haemolymph CYP 1B1 protein levels were increased after treatment with sublethal concentrations of atrazine. The increase in CYP 1B1 activities may be an adaptive mechanism to prevent harmful chemical accumulation or a reflection of the possibility of enhanced protection against atrazine toxicity. These results are in harmony with Londono et al. (2004) who found that exposing of Chironomus tentans to atrazine resulted in an increase in CYP 450 activity and total protein. Dong et al. (2009) stated that P450 content increased in male and female zebrafish Danio rerio after exposure to 0.01 mg/l atrazine. Omran and Salama (2013) reported that after sublethal Atrazine exposure, the level of CYP4501B1-like immunoreactivity increased in the hermaphrodite gland of Biomphalaria alexandrina.
Histological studies are increasingly being used as environmental stress indicators because they provide a specific biological endpoint of historical exposure and indicate the toxicant's direct action in the organs (Ramesh et al. 2018). Chronic exposure of male and female P. clarkii to sublethal concentration of atrazine (LC10 and LC25) exhibited severe gonad damage. Distorted architecture, spermatogonia distribution disturbance in some testicular acini, appearance of vacuoles in primary spermatocyte, lysis of some testicular acini, hyperplasia and tissues necrosis were observed in the treated P. clarkii testis. Separation of ovarian epithelium and oogenetic pouch, as well as lysis in connective tissue, was observed in the ovary of treated P. clarkii. These results were in harmony with Sheir et al. (2015) who reported that Trichoderma biofungicide, caused severe alterations in ovary and testis of Procambarus clarkii. In this context, Chandler et al. (2017) stated that atrazine caused degradation in tubular structure of testis of juvenile Cambarus bartonii. After 30 days of cypermethrin exposure, Srivastava et al. (2008) found that the gonad structure of Channa punctatus was altered, resulting in inflammation and vacuolization of the testis, as well as necrosis of testis tissues. These histological changes could be attributed to direct toxic effects of atrazine on the gonads.
In aquatic ecosystems, crayfish are considered a keystone species. In this study atrazine was shown to have severe effects on crayfish, which may have an impact on the role of crayfish as a regulator in local aquatic ecosystems. Understanding how atrazine affects crayfish could aid efforts to preserve the health of aquatic life. In the future, it will be useful to investigate the effect of atrazine on various life stages of crayfish by using new biomarkers.