Introduction

Terrestrial molluscs, snails and slugs are very important group that normally spread to different areas through human activities. Snails are regarded as pests due to their damage to cultivated crops as well as their role in carrying parasitic diseases affecting humans (Barker 2002; Hajian-Forooshani et al. 2020). Land snail, Monacha cartusiana and Eobania vermiculata (Muller 1773) are the most prevalent species in Egypt. They are recorded on clover, wheat, mango, orange, grapes and wood trees (Reham and Ramadane 2020). Land snails have shell which cover their soft body and allows the snails to survive under severe conditions of drought and heat (Crowell 1977). It is established that carbonic anhydrase speeds up the formation of biocarbonate, production of calcium carbonate and development of the shell (Wilbur and Jodrey 1955; Muller et al. 2013). The enzymes involved in the formation of the shell are phosphatase, phosphorylase and carbonic anhydrase (Digby 1968; Mobarak and Kandil 2014). It is difficult to control land snails because of their shells that protect them from any foreign compounds. Chemical molluscicide such as metaldehyde has strong effect on land snails. However, it cannot be used in moist places because treated snails quickly regain its moisture loss from their bodies and recover. Likewise, methomyl compound has negatively affected non-target species and increases environmental pollution (Mobarak and Kandil 2021; Mobarak et al. 2021). Therefore, alternative effective safe products should be tested against land snails. N- acetylcysteine (drug) is a sulfhydryl consisting of – compound and derived from amino acid L-cysteine. It is usually used to reduce the viscosity of mucus secretions and increase the ciliary clearance rate (Blackwell et al. 1996; Overveld et al. 2005; Tardiolo et al. 2018; Mobarak et al. 2021). Keeping in mind the above discussions, this study was designed to estimate the effect of acetylcysteine on the shells of two species of land snail; clover land snail, M. cartusiana and chocolate band snail, E. vermiculata, under laboratory and field conditions.

Materials and Methods

Experimental compound

Acetylcysteine (600 mg powder) was purchased from South Egypt drug Industries Company (Sedico) Egypt. The median lethal dose (LD₅₀) of acetylcysteine for rats is 5050 mg/kg (Golden 1971). Methomyl Lannate (90% Powder, Kafer El-Zayat Company, Egypt), is a carbamate insecticide compound recommended by Ministry of Agriculture and Land Reclamation (MALR) against land snail infestation in agriculture crops, at the rate of 8–10 kg/feddan. The LD₅₀ value for rats is 17–24 mg/kg.

Tested animals

Adult animals of the two species of land snails; M. cartusiana were obtained from clover field of Sumasta, Beni-Suef Governorate, Egypt, (N28°54’13 E30°54’36) and E. vermiculata, were collected from citrus trees at the nursery of Abu-Rawash district, Giza Governorate, Egypt, (N30°”8” E 31°. 5” 26”). Snails were transported to the Laboratory of the Harmful Animals Research Department, Sids Agriculture Research Station, Agriculture Research Center, (N28°54”21” E 30°57”12”). Snails of each species were put in plastic boxes having 8–10 cm moist soil, offered with fresh leaves of lettuce and covered with muslin cloth secured with rubber band to impede snail from escaping. Snails were acclimated for two weeks at 20 ± 2°C in the laboratory before beginning of the experiments.

Laboratory experiments

Thin film layer technique: The method of thin film layer was used according to Asher and Mirian (1981). Serial concentrations (0.15, 0.3, 0.6, 1.2, 1.8, 2.4 and 3.6%) of acetylcysteine were applied in Petri-dishes, for each of M. cartusiana and E. vermiculata individually. Two mL of each concentration of the compound was spread on the inner surface of each Petri-dish by moving the dish in circles. Water was evaporated in few minutes under room temperature leaving a thin film layer of the tested compound. A parallel control test was conducted using tap water only. The dead animals were daily counted and removed. Mortality percentages were calculated and LC₅₀ value was determined after seven days of treatment according to Finney (1971).

Biochemical studies: Each of the tested land snail species were treated individually with LC₂₅ of acetylcysteine for seven days to estimate the effect of acetylcysteine on carbonic anhydrase activity and the shell contents of calcium, phosphorus, magnesium and potassium.

Sample preparation

After seven days of treatment, the shell was removed from treated and untreated snail species. Then, the shell was grinded to determine the elements content of the shell. On the other hand, one gram of the snail soft tissue was homogenized under cooling for three minutes with 10 mL of sodium chloride 0.9 N, and then centrifuged (5000 rpm for 30 min). The resulting supernatant was used to determine the carbonic anhydrase activity.

Determination of carbonic anhydrase activity

The carbonic anhydrase activity was determined according to Barman (1974) using Novus Biologicals kits (USA). The developed color was measured at 450 nm using JENWAY 6305 UV/Vis Spectrophotometer.

Shell element content determination

Calcium (Ca) level determination: Calcium ion produces a blue color with methylthymol blue in an alkaline medium. The intensity of color is in proportion to the calcium concentration. The presence of hydroxyl 8-quinoline eliminates the interference due to the magnesium ions. The developed color was measured at 585 nm according to Gindler and King (1972) using Biodiagnostic (diagnostic and research reagents) kits purchased from Biodiagnostic Company, Egypt.

Phosphorus (P) level determination: Inorganic P present in shell solution as phosphate forms a phosphomolybdate complex with molybdic acid. The complex is reduced by stannous chloride to a blue color which can be measured calorimetrically at 640 nm according to El-Merzabani et al. (1977) using Biodiagnostic (diagnostic and research reagents) kits obtained from Biodiagnostic Company, Egypt.

Magnesium (Mg) level determination: Mg²⁺ react in an alkaline medium with the metallochromic dye calmagite to form a chromophore which absorbs at 520 nm according to Teitz (1983) using Biodiagnostic (Diagnostic and Research Reagents) kits purchased from Biodiagnostic Company, Egypt.

Potassium (K) level determination: Potassium ions in protein-free filtrate react with sodium tetraphenyl boron forming colloidal solution which can be measured calorimetrically at 420 nm according to Sunderman and Sunderman (1958) using Biodiagnostic (Diagnostic and Research Reagents) kits purchased from Biodiagnostic Company, Egypt.

Field experiments

Four plots (20 m² each) planted with clover and infected with M. cartusiana were chosen at Quftan, Sumsta district, Beni-Suef Governorate, Egypt, (N 28^ᵒ54’13 E30^ᵒ54’36). Another four plots planted with young citrus trees and infested with E. vermiculata were chosen at Abu-Rawash, Giza Governorate, Egypt, (N 30^ᵒ”8” E 31.5^ᵒ “26”). Other plots left without any treatment were taken as control. The most effective concentrations of acetylcysteine in the laboratory Table 1: LC₅₀ determination of acetycysteine against land snails, M. cartusiana and E. vermiculata, after one week of treatment using thin film layer technique

Concentration (%)	M. cartusiana		E. vermiculata
Concentration (%)	Mortality (%)	LC₅₀(%)	Mortality (%)	LC₅₀ (%)
0.15	0.0	0.6	0.0	1.5
0.3	10.0		0.0
0.6	40.0		10.0
1.2	80.0		30.0
1.8	100.0		60.0
2.4	100.0		90.0
3.6	100.0		100.0

Table 2: Effect of LC₂₅ of acetylcysteine on carbonic anhydrase (ng/mg) activity of land snails, M. cartusiana and E. vermiculata, after one week of treatment

Group	Carbonic anhydrase activity (ng/mg)
	M. cartusiana	E. vermiculata
Control	4.2 ± 0.23 ^a	5.10 ± 0.23 ^a
Treated	1.5 ± 0.12 ^b	2.47 ± 0.09 ^b
LSD	0.72	0.69

P ˂ 0.05

* Data are expressed as mean ± SE

* Means, which share the same superscript symbol(s), are not significantly different

Table 3: Effect of LC₂₅ on shell elements content of two land snails, M. cartusiana and E. vermiculata, after one week of treatment

Shell component	Species
	M. cartusiana			E. vermiculata
	Control	Treated	LSD	Control	Treated	LSD
Ca mg/g	24.1 ± 0.25^a	19.5 ± 0.28^b	1.1	21.5 ± 0.60^a	18.0 ± 0.46^b	2.1
P mg/ g	13.7 ± 0.08^b	15.1 ± 0.15^a	0.5	13.4 ± 0.25^b	14.9 ± 0.07^a	0.7
Mg mg/ g	0.67 ± 0.01^b	0.9 ± 0.01^a	0.02	0.55 ± 0.01^a	0.55 ± 0.01^a	-
K mmol /L	26.5 ± 0.28^a	27.5 ± 0.26^a	-	25.1 ± 0.10^a	23.5 ± 0.48^b	1.35

P ˂0.05

* Data are expressed as mean ± SE

* Means, which share the same superscript symbol(s), are not significantly different

tests were 1.8 and 3.6% for M. cartusiana and E. vermiculata, respectively. These concentrations were evaluated, in the field, against both land snail species and compared with methomyl 2% (MALR recommended compound) using spray method. Two replicates were applied for each treatment and others for control. A distance of ten meters was left between the plots. Survived snails were counted in each plot (in four corners and in center of each plot) pre and post treatment at 1, 3, 7, 15 and 21 days. The reduction in population of snails was calculated 21 days’ post treatment according to Henderson and Tilton (1952).

Statistical Analysis

Experimental design was completely randomized with different replicates. The results were statistically analyzed by one-way analysis of variance (ANOVA) and least significant difference (LSD) at (P < 0.05) using the COSTAT program (Glenn 2005).

Results

Laboratory studies: The efficacy of acetylcysteine

The results depicted that mortality percentage increased gradually with increasing acetylcysteine concentrations. However, the concentrations of 0.15, 0.3, 0.6, 1.2, 1.8, 2.4 and 3.6% presented 0.0, 10, 40, 80, 100, 100, and 100% mortality for M. cartusiana and 0.0, 0.0, 10.0, 30, 60, 90 and 100% for E. vermiculata, respectively. The LC₅₀ values were 0.6 and 1.5% for M. cartusiana and E. vermiculata, respectively after seven days of treatment (Table 1).

Biochemical studies: Carbonic anhydrase activity

The results revealed the effect of LC₂₅ of acetylcysteine on M. cartusiana, and E. vermiculata, after seven days of treatment. The results depicted that the activity of carbonic anhydrase decreased from 4.2 ng/mg in control to 1.5 ng/mg in treated M. cartusiana. It also showed the same trend in case of E. vermiculata, whereas it decreased from 5.10 ng/mg in control to 2.47 ng/mg in the treated snails. There were significant decreases in the enzymatic activities between control and treated snails (Table 2).

Effect of acetylcysteine on shell elements levels

The impacts of LC₂₅of acetylcysteine on shell levels of Ca, P, Mg and K are reported in Table 3 and Fig. 1–4. There were significant differences between treated and untreated snails. In the treated snails, Ca level decreased to 19.5 mg/g comparing with 24.1 mg/g in the control. Regarding P, Mg and K levels in M. cartusiana shell, values elevated to 15.1 mg/g, 0.9 mg/g and 27.5 mmol/L in treated snails compared to 13.7 mg/g, 0.67 mg/g and 25.5 mmol/L in the control, respectively. Concerning E. vermiculata, the Ca level decreased from 21.5 mg/g in control to 18 mg/g in treated snails and K level reduced from 25.1 mmol/L to 23.5 mmol/L. While P level was enhanced 14.9 mg/g in treated snails compared with 13.4 mg/g in the control. The level of Mg remained unchanged (0.55 mg/g) in both treated and untreated shells.

The field performance of acetylcysteine

The efficiency of acetylcysteine on M. cartusiana compared with methomyl after three weeks of application using spray technique was evaluated. The results indicated that the tested compound caused 94.7% reduction in snails’ population compared with 76.4% for methomyl. These results achieved significant reduction (P < 0.05) in M. cartusiana numbers (Table 4). Similar application against E. vermiculata, depicted that acetylcysteine achieved 90.1% reduction in snails´ population compared with methomyl which caused 74.9% reduction only. These results displayed significant reduction (P < 0.05) in snail numbers after treatment (Table 5).

Fig. 1: Untreated adult M. cartusiana

Fig. 2: Adult M. cartusiana treated with acetylcysteine showing broken shell

Fig. 3: Untreated adult E. vermiculata

Fig 4: Adult E. vermiculata treated with acetylcysteine showing color change of the shell

Discussion

The present study revealed the efficacy of acetylcysteine against M. cartusiana and E. vermiculata. The mortality percentages of both species were increased with increasing the compound concentrations. It may be due to the higher concentrations that reduced the ability of snails to repel the compound from their bodies by the mucus. The results also showed that M. cartusiana was more susceptible than E. vermiculata. These results may be attributed to the smaller size of M. cartusiana than E. vermiculata. Moreover, M. cartusiana secretes less mucus than E. vermiculata. Therefore, M. cartusiana is unable to excrete the compound from their bodies by mucus like E. vermiculata. Mucus is very important to snails and the mucus viscosity and production are changed post treatment (Livingstone et al. 1990; King and Rubin 2002). It was mentioned that 3.6% of acetylcysteine gave 100% mortality against M. cartusiana after seven days of treatment using bait technique (Mobarak et al. 2021).

In our study, treatment of both snail species with LC₂₅ of acetylcysteine produced noticeable decrease in carbonic anhydrase activity. These results may be attributed to the impact of acetylcysteine on hepatopancrease which inhibits the activity of carbonic anhydrase. It was clarified that carbonic anhydrase speeds up the formation of bicarbonate and the production of calcium carbonate to form the shell (Wilbur and Jodrey 1955; Muller et al. 2013). Also, the results may be attributed to reduction of shell calcium rate of treated animals that led to the shell became weaker and breakable. This perception agreed with Wilbur and Jodrey (1955). The mucus of animals is very thick and has high concentration of calcium contents (South 1992). Acetylcysteine decreased the snail mucus calcium rate and consequently, leading to reduction in mucus viscosity. This effect may be due to carbonic anhydrase inhibition post treatment. This finding is agreed with (Mobarak et al. 2021). The tested compound may inhibit the activity of the enzyme leading to prevention of calcium depositions. Previous study recorded that shell thinning due to decreasing the carbonic anhydrase activity by abamectin and thiamethoxam led to prevent calcium carbonate production and mortality of land snail, Theba pisana post treatment (El-Gendy et al. 2019). Other study showed a significant inhibition in the activity of carbonic anhydrase of the mussel of Mytilus galloprovincialis post treatment with cadmium (Lionetto et al. 2016).

The findings of the current study showed that M. cartusiana was more susceptible to acetylcysteine than E. vermiculata. This may be due to the smaller size and weaker shells of M. cartusiana compared to E. vermiculata. Moreover, the results revealed that Ca level was significantly decreased in the shell of both species after treatment. These results may be attributed to carbonic anhydrase inhibition which is responsible for deposition of calcium in shells. As reported by Mobarak and Kandil Table 4: Field application of acetylcysteine against land snail, M. cartusiana comparing with methomyl after three weeks of application as a spray technique

Treatment	Rate of application (g/L)	No. of snails survived pre-treatment		No. of snails survived post treatment		LSD	Reduction population (%)
Treatment	Rate of application (g/L)	No.	Mean ± SE	No.	Mean ± SE	LSD	Reduction population (%)
Acetylcysteine	18	248	24.8 ± 1.0 ^b	12	1.2 ± 0.5 ^d		94.7
Methomyl	20	388	38.8 ±1.2 ^a	84	8.4 ± 0.5 ^c		76.4
Control	-	314	31.4 ± 4.0 ^a	288	22.8 ± 2.9 ^b	6.9

P ˂ 0.05.

* Data are expressed as mean ± SE

* Means, which share the same superscript symbol(s), are not significantly different

Table 5: Field application of acetylcysteine against land snail, E. vermiculata comparing with methomyl after three weeks of application as a spray technique

Treatment	Rate of application (g/L)	No. of snails survived pre-treatment		No. of snails survived post treatment		LSD	Reduction population (%)
Treatment	Rate of application (g/L)	No.	Mean ± SE	No.	Mean ± SE	LSD	Reduction population (%)
Acetylcysteine	36	706	70.6 ± 8.9^a	64	6.4 ± 0.8^c		90.1
Methomyl	20	314	31.4 ± 1.9^b	72	7.2 ± 0.9^c		74.9
Control	-	704	70.4 ± 8.6^a	644	64.4 ± 7.7^a	17.6

P ˂ 0.05

* Data are expressed as mean ± SE

* Means, which share the same superscript symbol(s), are not significantly different

(2014), tannic acid reduced alkaline and acid phosphatase activities responsible for calcium participation in the shell of E. vermiculata and M. cartusiana. The results showed that P level increased in the shell of each tested snail species leading to reduction in the Ca level and reabsorbed Ca from the shell resulting in pathological changes. As mentioned in the previous study Taylor and Bushinsky (2009) that disturbances in the body level of P and Ca can lead to pathological changes. In addition, the Ca and Mg ions are known to have specific and opposite effect at the prejunctional nerve terminals of several cholinergic synapses (Jenkinson 1957). This finding supports our results, whereas the increased Mg level led to decreased Ca level causing weakness and breaking of shell of treated M. cartusiana. Similar results were reported in the eggshell in hens (Shastak and Rodehutscord 2015; Skrivan et al. 2016). The same trend occurred in case of K level, whereas its increase in the shell of M. cartusiana caused decreased Ca level which led to weakening of the shell of M. cartusiana. It was reported by Leach (1974) that K contents reduction in the hen causes egg shell thickness. While K level reduced in E. vermiculata compared to the control causing disturbance in shell component. Finally, treatment with acetylcysteine caused inhibition in the carbonic anhydrase responsible for the formation of the shell, consequently led to a decrease in the Ca level which led to shell weakness and making it fragile. It was investigated that acetylcysteine caused remarkable increase in alkaline phosphatase activity in M. cartusiana (Mobarak et al. 2021).

On the other side, the field results were in harmony with laboratory results, whereas it is proved that acetylcysteine was more effective against M. cartusiana than E. vermiculata. Moreover, the results confirmed that acetycysteine was more effective than methomyl against the two tested land snail species, as it caused high reduction percent in snail population. It may be due to the ability of acetylcysteine to weaken snail shells which enhanced penetration of the compound rapidly at the site of action. As recorded in our previous study, that acetylcysteine caused 95.0% reduction in snail numbers after three weeks of application using the bait method (Mobarak et al. 2021). Acetylsalicylic acid showed 33.4% reduction in M. obstructa population, while it caused 86.0% reduction in E. vermiculata population (Mobarak 2008). Chitosan achieved 74.3% population reduction of E. vermiculata after 21 days of treatment using the spray method under field conditions (Nada 2020).

Conclusion

From the previous findings, acetylcysteine proved to have strong effect on the shells of land snails leading to shell weakness and breakable, causing snail death. Therefore, it can be concluded that the compound could be used as an effective and safe molluscicide under Egyptian agricultural field conditions.

Acknowledgment

I wish to express my deepest gratitude to Dr. Aly El-Sherbiny Professor of Vertebrate Ecology, at the Harmful Animal Research Department, Plant Protection Research Institute, ARC, for correcting the manuscript’s language, and for scientific reviewing. Great and special thanks to Dr. Waheed Gabr, Professor at the Harmful Animal Research Department, Plant Protection Research Institute, ARC, for correcting the manuscript scientifically. We thank Dr. Ehab A. Abdallah, Senior Researcher at the Poultry Physiology and Director of the Physiology Lab, Poultry Department Unit, Animal Production Research Institute, Agriculture Research Center, Ministry of Agriculture, Egypt, for helping in evaluation of the elemental contents in the snail’s shell.

Author Contributions

Heba Y. Ahmed, Randa A. Kandil and Soha A. Mobarak proposed the research plan, processed the laboratory and field experiments and shared in writing the manuscript. All authors read and approved the final manuscript.

Consent for Publication

The authors´ consent for publication.

Funding Source

No fund, it is the personal effort of the author.

References

Asher RS, F Mirian (1981). The residual contact toxicity of Bay Sir 8514 to Spodoptera littoralis larva. Phytoparasitica 9:133‒137

Barker G (2002). Molluscs as crop pests. Landcare Research, Hamilton, New Zealand

Barman TE (1974). Enzyme Handbook, Vol. 1, p:9. Spring Velag. Berlin, Germany

Blackwell TS, TR Blackwell, EP Holden (1996). In vivo antioxidant treatment suppresses nuclear factor-kappa B activation and neutrophilic lung inflammation. J Immunol 157:1630‒1637

Crowell H (1977). Chemical Control of Terrestrial Slugs and Snails, p:4. Agriculture experiment station, Oregon State University, Corvallis, Oregon, USA

Digby PS (1968). The mechanism of calcification in the molluscan shell. In: Symposium of Zoological Society of London. Studies in the Structure, Physiology, and Ecology of Molluscs, pp:93–107. Academic Press, London

El-Gendy KS, MA Radwan, AF Gad, AE Khamis, EH Eshra (2019). Physiological trails of land snail, Theba pisana, as a simple endpoint to assess the exposure to some pollutants. Environ Sci Pollut Res 26:6422‒6430

El-Merzabani M, A El-Asaer, M Zakhary (1977). A new method for determination of inorganic phosphorus in serum without deporteinization. J Clin Chem Clin Biochem 15:715‒718

Finney DJ (1971). Probit Analysis, 3^rd edn. Cambridge University Press, Cambridge, UK

Gindler M, JD King (1972). Rapid clorimetric determination in biologic fluids with methomyl blue. Amer J Clin Pathol 58:376‒382

Glenn ND (2005). COHORT analysis, 2^nd edn. SAGe Publication, New Delhi, India

Golden E (1971). A compilulion of LD₅₀ value in newborn and adult animal. Toxicol Appl Pharmacol 18:185‒207

Hajian-Forooshani Z, J Vandermeer, I Perfecto (2020). Insights from excrement: Invasive gastropods shift diet to consume the coffee leaf rust and its mycoparasite. Ecology 101:1‒4

Henderson C, WF Tilton (1952). Tests with acaricides against the brown wheat mite. J Econ Entomol 18:157‒161

Jenkinson D (1957). The nature of the antagonism between calcium and magnesium ions at the neuromuscular junction. J Physiol 138:434‒444

King M, KB Rubin (2002). Pharmacological approaches to discovery and development of new mucolytic agents. Adv Drug Deliv Rev 54:1475‒1490

Leach R (1974). Studies on the potassium requirement of the laying hen. J Nutr 104:684‒686

Lionetto MG, R Caricato, ME Giordano, T Schettino (2016). Carbonic anhydrase based biomarkers: Potential application in human health and environmental sciences. Curr Biomark 6:40‒46

Livingstone CR, MA Andrews, SM Jenkins (1990). Model systems for the evaluation of mucolytic drugs: Acetylcysteine and S-carboxymethylcysteine. J Pharm 42:73‒78

Mobarak SA (2008). Efficacy and toxicological studies for some binary mixture of some pesticides with acetylsalicylic or tanic acids against the land snails. Faculty of Agriculture, Cairo University, Egypt

Mobarak SA, RA Kandil (2021). Sever stress of Cuprol compound against land snail respiration. Plant Cell Biotechnol Mol Biol 22:246‒253

Mobarak SA, RA Kandil (2014). Efficacy of different compounds against the principle calcium precipitation parameters of terrestrial snails. Acad J Biol Sci 6:1‒10

Mobarak SA, HY Ahmed, RA Kandil (2021). Efficiency of Acetylcysteine (drug) as a molluscicide. Egypt Acad J Biol Sci 13:147‒155

Muller OF (1773). Vermivm terrestrium et fluviatilium, sen animalium infusoriorum, helminthicorum et testaceorum, non marinorum, succincta historia, Vol. 2, p:214. Testacea. Havnie et Lipsiae

Muller WE, HC Schroder, U Schlossmacher, M Neufurth, W Geurtsen, M Korzhev, X Wang (2013). The enzyme carbonic anhydrase as an integral component of biogenic Ca-carbonate formation in sponge spicules. FEBS Open Biol 3:357‒362

Nada MA (2020). Efficacy of certain chemical control agents against land snail special references to their physiological effects. Ph.D. Thesis, p:146. Faculty of Agriculture, Cairo University, Egypt

Overveld FJV, U Demkow, D Gorecka (2005). New developments in the treatment of COPD: Comparing the effects of inhaled corticosteroids and Nacetylcysteine. J Physiol Pharmacol 56:135‒142

Reham F, R Ramadane (2020). Taxonomic Key as a simple tool for identifying and determining the abundant terrestrial snails in Egyptian fields. Egypt Acad J Biol Sci 12:173‒203

Shastak Y, M Rodehutcord (2015). A review of the role of magnesium in poultry nutrition. World Poult Sci 71:125‒137

Skrivan M, M Englmaierova, V Marounek, T Skrivanova, T Taubner (2016). Effect of dietary magnesium, calcium, phosphorus, and limestone grain size on productive performance and eggshell quality of hens. Czech J Anim Sci 61:473‒480

South A (1992). Terrestrial slug, p:428. Chapman & Hall, London

Sunderman FWJ, FW Sunderman (1958). Studies in serum electrolytes. 22.A rapid, reliable method for serum potassium using tetraphenylboron. Amer J Clin Pathol 29:95‒103

Tardiolo G, P Bramanti, E Mazzon (2018). Overview on the effects of N-acetylcysteine in neurodegenerative diseases. Molecule 23:1‒20

Taylor J, D Bushinsky (2009). Calcium and phosphorus Homeostasis. Blood Purif 27:387–394

Teitz NW (1983). Clinical guide to laboratory Tests 95, p:52. W.B. Saunders, Philadelphia, Pennsylvania, USA

Wilbur K, L Jodrey (1955). Studies on Shell formation. In: The inhibition of shell formation by carbonic anhydrase inhibitors, Vol. 117, p:569. Div John Wiley & Sons Inc., New York, USA