International Journal of Environmental Studies ISSN: 0020-7233 (Print) 1029-0400 (Online) Journal homepage: https://www.tandfonline.com/loi/genv20 Reproductive and developmental alterations in Biomphalaria alexandrina snails exposed to three phenolic compounds Wafaa S.F. Hasheesh, Haggag A. Mohamed, Sara S.M. Sayed & Rasha M.F. Abu El-Khair To cite this article: Wafaa S.F. Hasheesh, Haggag A. Mohamed, Sara S.M. Sayed & Rasha M.F. Abu El-Khair (2019): Reproductive and developmental alterations in Biomphalariaalexandrina snails exposed to three phenolic compounds, International Journal of Environmental Studies To link to this article: https://doi.org/10.1080/00207233.2019.1686908 Published online: 06 Nov 2019. Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=genv20 INTERNATIONAL JOURNAL OF ENVIRONMENTAL STUDIES https://doi.org/10.1080/00207233.2019.1686908 ARTICLE Reproductive and developmental alterations in Biomphalaria alexandrina snails exposed to three phenolic compounds Wafaa S.F. Hasheesha, Haggag A. Mohameda, Sara S.M. Sayed b and Rasha M.F. Abu El-Khaira aZoology Department, Faculty of Science, Cairo University, Giza, Egypt; bEnvironmental Research and Medical Malacology Department, Theodor Bilharz Research Institute, Giza, Egypt ABSTRACT KEYWORDS The present work aims to assess the reprotoxicity in Biomphalaria Salicylic acid; benzoic acid; alexandrina snails and developmental and hatchability alterations of pyrogallol; reproduction their eggs after exposure to three phenolic compounds, viz., salicylic acid, benzoic acid and pyrogallol. The half lethal concentrations (LC50) of the three tested compounds were 574, 674 and 350 mg L−1, respectively after 24 h. The tested concentrations of all compounds showed a harmful effect on development and hatchability of eggs regardless of the age of embryos. Exposure of snails to sub-lethal concentrations of salicylic acid resulted in a significant reduction in their survival rate, and ½ LC50 of both benzoic acid and pyrogallol led to a sudden death of snails after the 1st week of the experiment. Therefore, the egg laying capacity showed a noticeable impairment throughout the whole experimental period. Moreover, the hermaph- rodite glands of treated snails have mirrored the deleterious effects that occurred in the total reproductive rate. Introduction Phenolic compounds have been listed by the United States Environmental Protection Agency (USEPA) and the European Union (EU) as pollutants of priority concern because of their acute toxicity and severe long-term effects on human beings and animals [1]. The biggest contribution to environmental pollution with respect to pharmaceuticals comes from the active substances of these drugs or their metabolites excreted from the body of both human and animal patients via urine or faeces [2]. Apart from daily common uses, salicylic acid, benzoic acid and pyrogallol are used by several industries and are discharged into the aquatic environment. Little information has been recorded about the chronic toxicity of these phenolic compounds to some aquatic organisms. Salicylic acid is a non-steroidal anti-inflammatory drug, which represents the most commonly found contaminant in the water environment [3]. Some of these drugs (mainly diclofenac and ibuprofen) exhibit an increasing hazardous effect on aquatic ecosystem [4]. On the other hand, salicylic acid acts as a plant growth regulator, a signalling molecule for CONTACT Sara S.M. Sayed [email protected] Department of Environmental Research and Medical Malacology,Theodor Bilharz Research Institute, Giza 12411, Egypt © 2019 Informa UK Limited, trading as Taylor & Francis Group Published online 06 Nov 2019 2 W. S. F. HASHEESH ET AL. defence response in plants against pathogen attack [5]. It is commonly used in chemical peeling treatments of several skin disorders [6]. Benzoic acid is commonly used for preserving foods, fats and fruit juices, also for alkaloid solutions and curing tobacco, besides being applied as a herbicide [7], and as an antifungal agent in pharmaceutical industries [8,9]. It is frequently found in the effluents of coal refining, paper and pulp mill, and in agricultural runoff [10]. Pyrogallol was the first synthetic organic dye used on human hair and then used as a modifier in the oxidation of dyes [11]. Afterwards, it was used as an active reducer of gold, silver, and mercury salts [12], and in the manufacture of pharma- ceuticals and pesticides [13]. Medically, pyrogallol has been used as a topical antipsoriatic [14]. Furthermore, it is used as an anti-algal compound and has been confirmed to exhibit one of the most intensive inhibitory effects on Microcystis aeruginosa [15]andChlorella Pyrenoidosa [16]. Few studies have examined the effects of different pharmaceuticals on aquatic inverte- brates. Disturbances in the early development frequently lead to arrested development and inviability. Accordingly, procedures and protocols implemented for toxicity and developmental bioassays should be applied in ecotoxicological studies [17]. Biomphalaria alexandrina snails are important aquatic organisms dwelling in rivers, ponds and lakes in Egypt. Toxicological assays employing Biomphalaria species have shown their value as biomarkers of aquatic toxicity, and they are considered as good indicators for biomoni- toring studies [18]. Therefore, the present study aims to assess the effect of salicylic acid, benzoic acid and pyrogallol on survival rate and reproduction of B. alexandrina snails, besides development and hatchability of their eggs. Materials and methods Experimental materials Salicylic acid (C7H6O3) is a colourless crystalline powder (CAS Number: 69-72-7), benzoic acid (C7H6O2) is a colourless crystalline powder (CAS Number: 65-85-0) and pyrogallol (C6H6O3) is a white water-soluble powder (CAS Number: 87-66-1).They were purchased from Cornell LAB Company, Cairo, Egypt. Bioassay tests Serial concentrations of pyrogallol were directly prepared in 1000 ml of dechlorinated tap water. Both salicylic acid and benzoic acid powders (insoluble in water), were firstly dissolved in 500 µl of 0.5% dimethyl sulphoxide (DMSO), then 1000 ml of dechlorinated tap water was added. The final concentrations of DMSO in the solutions were less than 0.001%. Therefore, its effect was excluded as it has no effect on snails in its minor concentration [19]. Three replicates, each of 10 snails/L (8–10 mm shell diameter) were exposed to each concentration of the three tested compounds for 24 h. Another group of snails was maintained in dechlorinated tap water as a control group. At the end of the exposure period, snails were removed from each tested concentration, washed thoroughly with dechlorinated tap water and transferred to another container for a recovery period of 24 h. Then, dead snails were counted, and LC50 and LC90 concentrations were computed using SPSS version 17.0 (SPSS, Inc., Chicago, IL) for windows. INTERNATIONAL JOURNAL OF ENVIRONMENTAL STUDIES 3 Survival rate and egg laying capacity of snails 1 Adult B. alexandrina snails were exposed to sub-lethal concentrations ( /10 LC50,¼LC50 and ½ LC50) of each tested phenolic compound, separately. Three replicates were prepared from each concentration, each of 10 snails/L. Another group of snails was maintained in clean water as a control group. Snails were fed daily with dried lettuce leaves. The survival rate was recorded, and egg masses laid by the exposed and control snails were weekly collected and counted using a dissecting microscope [20]. Histopathological examination – Healthy adult snails (8 10 mm shell diameter) were exposed to ¼ LC50 (143.5, 168.5 and 87.5 ppm) of salicylic acid, benzoic acid and pyrogallol, respectively for four successive weeks. A group of snails was maintained in dechlorinated tap water as a control group. Thereafter, snails of treated and control groups were washed with water then dried. The shells were gently crushed and the hermaphrodite gland of each snail was gently separated then fixed in Bouin’s solution, dehydrated using ascending grades of ethanol, embedded in paraffinwaxandfinally sectioned at 6µ. Sections were stained with HaematoxylinandEosinstain,thenmicroscopi- cally examined and photographed by a Zeiss Video camera™,Germany. Development and hatchability of eggs About 100 embryos of each egg mass one-day and three-day old were collected separately. Three replicates of each group of egg masses were exposed to different concentrations of the three tested phenolic compounds. Unexposed one and three days-old egg masses were maintained in dechlorinated water as control groups. After 24 h, treated and control egg masses were transferred to clean water till hatching and the percentage of hatchability was calculated. During the experiment, egg masses were microscopically examined and photo- graphed to record the egg development [17]. Statistical analysis The hatchability percentage of eggs and the survival rate of snails were analysed by Chi- square values of contingency tables using the statistical programme SPSS version 17 (SPSS, Inc., Chicago, IL) for windows. Results Toxicity of tested compounds Pyrogallol was more toxic to Biomphalaria alexandrina snails than salicylic acid and −1 benzoic acid, as LC50 values were 350, 574 and 674 mg L respectively (Table 1). Survival rate and egg laying capacity of snails Generally, the survival rates of all treated groups were less than those of the control group – 1 (Figure 1(a c)). The survival rate of snails exposed to /10 LC50 of salicylic acid showed 4 W. S. F. HASHEESH ET AL. Table 1. Probit analysis for toxic effect of the three phenolic compounds on adult B. alexandrina snails after 24 h of exposure. Concentration (mg L−1) Confidence limit 1 Phenolic compound LC50 slope of LC50 LC90 /10 LC50 ¼LC50 ½LC50 Salicylic acid 574 1.24 511.7–643.6 730 57.4 143.5 287 Benzoic acid 674 1.13 614.6–769.3 780 67.4 168.5 337 Pyrogallol 350 1.27 301.5–398.5 454 35.0 87.5 175 a gradual reduction reaching 60% at the 4th week of exposure, followed by a long steady period started from the 5th week till the end of the exposure period.