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Arch. Environ. Contain. Toxicol. 21, 35%364 (1991)

9 1991 Spfinger-Verlag New York Inc.

Toxicity Evaluation of Ammonium Sulphate and Urea to Three Developmental Stages of Freshwater Snails P. B. Tchounwou*, A. J. Englande, Jr.*, and E. A. Malek** *Department of Environmental Health Sciences and **Department of Tropical Medicine and Parasitology, School of Public Health and Tropical Medicine, Tulane University Medical Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112, USA Abstract, Studies were performed to evaluate the toxic effects of ammonium sulphate and urea (chemical fertilizers currently applied in ricelands of Cameroon) against eggs, juveniles, and adults of two species of freshwater snails (Helisoma trivolvis and Biomphalaria havanensis). Results obtained from ammonium sulphate tests indicated 24-h LCso values of 558 mg/L and 669 mg/L for eggs; 393 mg/L and 526 mg/L for juveniles, and 701 mg/L and 657 mg/L for adults of H. trivolvis and B. havanensis, respectively. Similar analysis with urea revealed LCso values of 14,241 mg/L and 13,532 mg/L for eggs; 18,255 mg/L and 24,504 mg/L for juveniles and 30,060 mg/L and 26,024 mg/L for adults of H. trivolvis and B. havanensis, respectively. Following 48 h exposure, the concentrations of ammonium sulphate killing 100% of snails were 1,250 mg/L and 1,000 mg/L for the adults of H. trivolvis and of B. havanensis, respectively. Those of urea were computed to be 25,000 mg/L for H. trivolvis and 35,000 mg/L for B. havanensis. In rice culture in Cameroon, these fertilizers are applied at doses of 100 kg/ha (ammonium sulphate) and of 150 kg/ha (urea); hence, the above found concentrations lethal to snails appeared to be 10 to 13 times (ammonium sulphate) and to be 165 to 235 times (urea) higher assuming an average water depth of 10 cm in these ricefields. Therefore, the use of ammonium sulphate and urea as chemical fertilizers in ricelands of the Republic of Cameroon might adversely affect the survival of freshwater snails only in the case of spills or of stressful environmental conditions. Under normal laboratory conditions, both chemicals show a low molluscicidal activity with urea being about 25 to 35 times less potent than ammonium sulphate.

The use of chemical fertilizers throughout the world has increased strikingly following World War II along with the restoration and expansion of manufacturing capacity. In tropical ecosystems the primary use of fertilizers is to produce a significant increase in crop yield. Thus, most research concerning fertilizers has been based on productivity evaluation for increasing agricultural crop yield. However, Hamdy et al. (1984) recently investigated the ovicidal and larvicidal

properties of some tropical fertilizers against Ascaris suum eggs, Hymenolepis nana eggs and Ancylostoma duodenale larvae. These fertilizers included ammonium sulphate, ammonium nitrate, ammonium carbonate, urea, potassium chloride and potassium carbonate. The authors concluded that the use of these chemicals for agricultural purposes is expected to assist in the control of ancylostomiasis and hymenolopiasis. In the ricelands of the Republic of Cameroon, especially in the Yagoua area, most heavily applied chemicals are nitrogen-based fertilizers. Recent information given by SEMRY; the q u a s i - p u b l i c c o m p a n y m a n a g i n g the r i c e l a n d s of Cameroon indicates that ammonium sulphate and urea are being applied twice (during the two growing seasons) annually. Doses of 100 kg/ha for ammonium sulphate and of 150 kg/ha for urea are commonly employed. The literature on schistosomiasis research is moot regarding the effects of such plant fertilizers on the survival of larval stages and intermediate hosts of human schistosomes.

Materials and Methods Materials Ammonium sulphate, [(NH4)2804]; MW 132.13; stock # A-4915; lot # 65F-0263 was purchased from Sigma Co. Urea, NH2CONHz; MW 60.06; stock # U-5128; lot # 96F-0582 was also obtained from Sigma Co. For each of the chemicals tested, a stock solution was prepared and subsequently diluted for desired test concentrations of the chemical. A 10% stock solution of ammonium sulphate or urea were made by dissolving 10 grams of either chemical with Kentwood Spring water (commercialized well water) in 100 ml solution. Due to the lack of availabilityof susceptible snails from Cameroon Helisoma trivolvis from Louisiana and Biomphalaria havanensis from the Dominican Republic were used as test organisms.

Methods The experimental protocol described in detail in the companion paper dealing with toxicity evaluation of selected pesticides to freshwater snails was applied in this study.

Table 1. Toxicity of ammonium sulphate to H. trivolvis snails

Table 2. Toxicity of ammonium sulphate to B. havanensis snails

95% confidence limits

Stage of snail

Toxicity parameter

Concentration (mg/L)

429.83 831.76 1868.24 10.14

Eggs a

LC 5 LCs0 LC95 SLOPE*

429.63 669.19 1,042.31 8.55

359.06 623.11 941.62 6.38

481.37 715.10 1219.96 10.71

132.66 338.57 696.04 3.81

235.07 444.28 1042.45 6.53

Juveniles a (2-3 mm)

LC 5 LCso LC95 SLOPE*

332.00 526.44 834.77 8.22

238.57 462.74 728.66 5.27

394.46 584.53 1069.63 11.16

473.31 801.29 1356.54 7.19

373.62 727.76 1205.83 5.32

548.49 870.99 1626.00 9.06

Adults a (5-6 ram)

LC 5 LCso LC95 SLOPE*

361.94 658.90 1199.48 6.32

238.57 566.32 1015.87 4.11

447.26 743.77 1624.72 8.54

460.35 700.73 1066.62 9.01

344.68 624.09 940.14 5.84

535.94 772.56 1338.55 12.19

Adults b (5-6 mm)

LC 5 LCs0 LC95 SLOPE*

240.56 490.79 1001.30 5.31

151.91 413.82 827.45 3.52

307.04 563.08 t406.80 7.10

Stage of snail

Toxicity parameter

Concentration (mg/L)

Eggs a

LC5 LCso LC95 SLOPE*

282.42 557.64 1101.06 5.57

7.51 258.66 767.41 0.99

Juveniles a (3-5 mm)

LC5 LCso LC95 SLOPE*

188.92 392.94 817.25 5.17

Adults a (8-10 mm)

LCs LCso LC95 SLOPE*

Adults b (8-10 mm)

LC5 LCso LC95 SLOPE*

Lower

Upper

N

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Upper

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a 24 hours exposure b 48 hours exposure * : Slope in mg/1-1

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95% confidence limits

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1. Concentration--effect curves for !000 Fig. ammonium sulphate against H. trivolvis eggs, juveniles and adults

Table 3. Toxicity of urea to H. trivolvis snails

Stage of snail

Toxicity parameter

Concentration (rag/L)

Eggs a

LC5 LCso LC95 SLOPE*

Juveniles a (3-5 mm)

Table 4. Toxicity of urea to B. havanensis snails 95%confidence limits

Stage of snail

Toxicity parameter

Concentration (rag/L)

11988.17 17229.32 53192.58 17.37

Eggsa

LC5 LCso LC95 SLOPE*

11513.22 17389.45 23798,38 8.52

14062.12 19129.52 28719.96 13.75

Juveniles" (2-3 ram)

17594.53 22997.59 30059.66 14.14

15462.66 21959.91 28358.79 10.23

18994.86 23887.75 33037.14 18.06

8813.03 13476.59 20607.96 8.92

6529.76 11957.82 18128.32 5.73

10299.30 14888,23 25997.23 12.10

Lower

Upper

9790.66 14240.75 20714.30 10,11

3425.46 11235.35 17154.91 2.84

LC5 LCso LC95 SLOPE*

12989.43 18253.51 25650.86 11.13

Adults a (8-10 mm)

LC5 LCso LC95 SLOPE*

Adults b (8-10 ram)

LC 5 LCso LC95 SLOPE*

24 hours exposure b 48 hours exposure * : Slope in mg/1-1

95% confidence limits Lower

Upper

10903.05 13532.13 16707.19 17.53

9934.68 13003.22 15951.14 13.01

11565.51 14036,68 18176.67 22.05

LC 5 LCso LC95 SLOPE*

18019.83 24504.37 33322,46 12.32

14984.44 22795.82 30473.18 8.23

19935.88 26148.72 39031.82 16.41

Adults a (5-6 mm)

LC5 LCs0 LC95 SLOPE*

18211.15 26024.16 37189.09 10.61

14798.57 24139.99 33438.05 7.01

20358.14 27965,52 45239.63 14.29

Adults b (5-6 ram)

LC 5 LCso LC95 SLOPE*

12391.80 21412.08 36998.48 6.93

9284.16 19269.66 32957.28 4.79

14591.80 23538.43 47372.48 9.06

a 24 hours exposure b 48 hours exposure * : Slope in mg/1-1

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Fig. 2. Concentration-effect curves for ammonium sulphate against B. havanensis eggs, juveniles and adults

362

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Fig. 3. Concentration-effect curves for urea against H. trivolvis eggs, juveniles and adults

be 1,067 mg/L for the adults of H. trivolvis (Table 1) and to be 1,001 mg/L for those of B. havanensis (Table 2).

Toxicological Studies with A m m o n i u m Sulphate Toxicological Studies with Urea Tables 1 and 2 present the concentration-effect parameters obtained from a computer analysis (US EPA Probit Analysis Program Version 1.3) of the results of the studies designed to evaluate the toxicity of ammonium sulphate to H. trivolvis and B. havanensis snails. The 24-h LCso values were 558 mg/L, 393 mg/L and 801 mg/L for eggs, juveniles and adults of H. trivolvis, respectively (Table 1). The 24-h LCso concentrations for eggs, juveniles and adults of B. havanensis were observed at 669 rag/L, 526 mg/L and 659 mg/L, respectively (Table 2). LCso values obtained following 48-hour exposures were 701 mg/L and 491 mg/L for adults of H. trivolvis and B. havanensis respectively. The LC95 values were calculated to

The concentration-effect parameters results obtained from the analysis of toxicity test data for urea against H. trivolvis and B. havanensis snails are presented in Tables 3 and 4. The 24-hour LCso values were found to be 14,241 mg/L, 18,254 mg/L and 22,998 mg/L for eggs, juveniles and adults of H. trivolvis respectively, (Table 3); and to be 13,532 mg/L, 24,504 mg/L and 26,024 mg/L for eggs, juveniles and adults of B. havanensis, respectively (Table 4). Following 48 h of exposure, the LCso values were computed as 13,477 mg/L and 21,412 mg/L for the adults of Helisoma and of Biomphalaria. A 48-h exposure resulted in LC95 values of 20,608 mg/L and 21,412 mg/L for the adults of H. trivolvis and of B. havanensis, respectively.

Toxicity of Ammonium Sulphate and Urea to Snails

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Discussion

Tests with A m m o n i u m S u l p h a t e

Based on the 24-h bioassay results, H. trivolvis and B. havanensis snails presented similar susceptibilities to ammonium sulphate toxicity. However, results obtained following 48 h exposure indicated H. trivolvis to be less susceptible than B. havanensis. The eggs appeared to be less susceptible to ammonium sulphate toxicity than the juveniles for both species of snails as shown in Figures 1 and 2. This effect was more apparent with B. havanensis where similar susceptibilities were noticed for both eggs and adults (Figure 2). Similar results have also been reported by H o p f and Muller (1962) and by Ritchie et al. (1963). Thus, a chemical may be less t o x i c to eggs but lethal to the subsequent emerging snails. Such an effect has also been observed with mosquitoe eggs. This phenomenon may be due to the fact that snail eggs are surrounded by an envelope which forms a protective barrier.

50000

100000

Fig. 4. Concentration-effect curves for

urea against B. havanensis eggs, juveniles and adults

The penetration of a chemical compound through this envelope will depend on several factors including the physical and chemical properties of the compound itself. The observations on snail behavior during this study revealed that the mortalities of snails in ammonium sulphate solutions could be attributed to excessive bleeding. During preliminary screenings for range findings, it was found that a 10% solution of ammonium sulphate killed snails on contact, producing heavy hemorrhages. In 1% solution, snails retracted within their shells and bleeding started to occur after 1-2 h post-exposure. Rapid mortalities of snails in high concentrations of ammonium sulphate could be attributed to the acidity of these solutions. F o r example, the p H ' s of 3% and 5% solutions of ammonium sulphate were 6.4 and 5.1, respectively. This increase in acidity with increase in ammonium sulphate concentration may be attributed to the formation of sulphuric acid (H2SO4) as the fertilizer dissolves in water. In very dilute solutions however, the toxicity of ammonium sulphate

364 may be attributed to the formation of un-ionized ammonia. Data obtained indicated that the toxicity of ammonium sulphate was highly influenced by changes in environmental parameters, with the pH being the most important.

P.B. Tchounwou et al. effect or the length of time for an effect to be manifested. For example, a 10% solution of urea could cause bleeding to snails after 1.5 h post-exposure; while a 10% solution of ammonium sulphate resulted in heavy bleeding and death upon contact.

Tests with Urea Figures 3 and 4 show the concentration-effect curves obtained from the bioassays evaluating urea effects. As illustrated, H. trivolvis and B. havanensis snails displayed similar susceptibilities to urea toxicity. The eggs appeared to be more sensitive to urea toxicity than the juveniles which also showed a higher susceptibility than the adults. Reduced amounts of urea were required for a given percent kill during longer exposure times as expected. The concentrations of urea affecting snail mortality during 24 h and 48 h of exposure were hundreds of times higher than the field doses typically applied for increasing crop yield. Thus, from a practical standpoint urea possesses a very low molluscicidal activity. Under the current agricultural practices, this chemical should not affect the survival of freshwater snails except perhaps under spill or overdosing situations. However, urea has been suggested to be used in conjunction with chemical molluscicides. Perret et al. (1972) studied the influence of urea on the molluscicidal activity of N-tritylmorpholine and pointed out that urea possesses some dispersive properties. When diluted with water, the endothermic reaction of urea caused sufficient movements to thoroughly disperse the molluscicide. On the other hand, urea as a nutritive substance, may increase the biomass of plankton in such quantities that snail breeding may be inhibited. Snail behavior in urea solutions was similar to that observed in ammonium sulphate solutions. The only difference detected were the concentration level needed to produce an

Acknowledgments. We thank Dr. Barnett L. Cline, Chairman of the Department of Tropical Medicine and Parasitology for his helpful suggestions throughout this work; and Dr. Ernesto Ruiz-Tiben, Epidemiologist at the Center for Disease Control for reviewing and editing the manuscript. This research was financially supported by the USAID-Cameroon: Health Constraints to Rural Development Project.

References Hamdy EI, Ahmed THE, Amin FMA, EL-Matarawy OM, E1Rahimy HM (1984) Laboratory studies on the ovicidal and larvicidal effects of some artificial fertilizers. J Egypt Soc Parasitol 14(1):131-136 Hopf H S, Muller LR (1962) Laboratory breeding and testing of Australorbis glabratus for molluscicide screening. Bull Wld Hlth Org 27:783-789 Perret P, Egger M, Degremont AA (1972) Essai de lutte antimollusque par augmentation de la biomasse planctonique et traitement molluscicide: Association Urea-N-Tritylorpholine. Acta Tropica 29(2):175-181 Ritchie LS, Frick LP, Berrois-Duran LA, Fox I (1963) Molluscicidal qualities of sodium pentachlorophenate (NaPCP) revealed by 6-hour and 24-hour exposures against representative stages and sizes of Australorbis glabratus. Bull Wld Hlth Org 29:421-427

Manuscript received November 24, 1990 and in revised form June 4, 1991.