The effect of ammonium nitrate fertiliser on frog (Rana temporaria) survival

Agriculture Ecosystems & Envtronment ELSEVIER

Agriculture, Ecosystemsand Environment61 (1997) 69-74

Short communication

The effect of ammonium nitrate fertiliser on frog ( Rana temporaria) survival R.S. Oldham a,* , D.M. Latham a, D. Hilton-Brown a, M. Towns a, A.S. Cooke b, A. Burn b a Department of Biological Sciences, De Montfort University, Leicester LE7 9SU, UK b English Nature, NorthminsterHouse, PeterboroughPEI 1UA, UK

Accepted 17 June 1996

Abstract

The toxicity of ammonium nitrate fertiliser to common frogs (Rana temporaria) was tested in the laboratory and field. Granular ammonium nitrate is the most commonly used fertiliser in Britain, especially during the spring, when adult frogs migrate over land. Ammonium nitrate was acutely toxic to frogs at concentrations well below those recommended for field application. However, it lost its acute effect when dissolved in the soil and even on relatively dry soil (7% moisture), granules dissolved in less than 3 hours. A potentially high mortality rate owing to ammonium nitrate is probably mitigated by the fortuitous asynchrony between fertiliser application during daylight, and frog migration at night. It remains to be determined whether there are sublethal effects and whether fertilisers that dissolve more slowly are implicated in the widespread amphibian declines in agricultural areas observed since the Second World War. Keywords: Ammoniumnitrate; Feniliser; Rana temporaria; Frog

1. Introduction

In Britain, the common frog (Rana temporaria) has undergone a major decline in agricultural areas since the Second World War (Cooke, 1972; Cooke and Scorgie, 1983) generally attributed to agricultural development. Whilst other agricultural factors probably play a part, there is a significant negative correlation (Rs = - 0 . 8 8 6 ) on a regional basis, between the percentage of farmland receiving nitrogen fertiliser during the period 1980-1990 (Chalmers et

Corresponding author.

al., 1990) and the index of change in frog status during the same period, as defined by Hilton-Brown and Oldham (Hilton-Brown and Oldham, 1991). During this period frog status has declined or shown little change in regions with the highest fertiliser application levels (Oldham et al., 1993). This indirect evidence of a relationship is augmented by field observations in Denmark of the acute toxicity of agricultural fertilisers to Rana dalrnatina (K. Fog, personal communication, 1989). There is a possibility that the widespread use of fertiliser may be a contributory factor to frog declines in the UK. Three major changes in fertiliser use have occurred during the period since the Second World War. First, the use of nitrogen based feniliser in-

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R.S. Oldham et al. / Agriculture, Ecosystems and Environment 61 (1997) 69-74

creased five-fold (Agricultural Development and Advisory Service, 1992). By 1988 average total nitrogen application levels had stabilised at 169 kg h a for arable, 178 kg ha -~ for dairying and 116 kg ha-~ for pasture. Secondly use of straight nitrate fertilisers (particularly ammonium nitrate), on all farm types increased from an estimated 23 kg h a in 1969 to 125 kg h a - I in 1988, whilst the use of compound mixes (nitrogen combined with other nutrients) fell from 61 kg h a - ~ to 24 kg ha- ~(Chaimers et al., 1990). Thirdly there has been a more recent shift towards the spring in the peak period of fertiliser application (Chalmers et al., 1990). In 1985, 17% of total compound nitrogen was applied after January; this rose to 77% by 1990. Application of straight nitrates is also now concentrated during the spring months; 67% of ammonium nitrate is applied between March and April. Frogs commonly migrate over agricultural land on their way to the breeding site during the spring and so risk exposure to fertiliser if this movement coincides with a recent application. Ammonium nitrate is the most frequently used fertiliser and this study investigated its possible role in contributing to the decline in frog populations. We examined the relationship between the application levels of ammonium nitrate and its acute toxicity to frogs. In this study we concentrated on effects on adult frogs and in order to reduce variability we restricted the experiments to males. Three sets of experiments were performed using ammonium nitrate, the first two to test for acute toxicity, one in the laboratory, the other in the field under the conditions of standard agricultural practice, and a third, in the laboratory, to test for the persistence of toxic effects.

2. Methods 2.1. Laboratory trials on acute toxicity of ammonium nitrate to adult frogs Two substrates were used in the trials, moist chromatography paper to enable precise control over moisture content and degree of exposure to granules, and soil to ensure closer approximation to realistic field conditions. Granules were fixed to moist chromatography paper or spread on soil (29% moisture

content) on the base of a 30 × 15 X 15 cm aquarium. The national average ammonium nitrate application rate is 12-18 g m -2 (Agricultural Development and Advisory Service, 1992), but the recommended rate for farms in the Scraptoft area of Leicestershire is 49.4 g m -2 (C. Draper, personal communication, 1991). On each substrate, frogs were exposed to four concentrations (fractions of 49.4 g m -2) from 1.5 g m -2 ( 1 / 3 2 ; approximately 1 granule per 8 cm 2) up to a maximum of 28.4 g m -2 ( 1 / 2 ; 1 granule per cm2). Animals were kept, singly, at temperatures between 18° and 21°C under continuous observation, with readings of buccal and lung ventilation taken every 5 min, for the first hour, followed by hourly observations for a further 5 h and a final observation 24 h after the start of exposure. Additional readings were taken at any stage when the condition of a frog was thought to warrant it. Exposure for longer than 24 h was not considered representative of possible field conditions for reasons explained below. Frogs were then washed in running water and kept under observation for 1 week, after which they were released. Each trial involving exposure to fertiliser was paralleled by a control on an identical substrate. If frogs exhibited symptoms of acute toxicity, recognised either through characteristic changes in ventilation rates or aberrant behaviour they were immediately removed from the unit and washed thoroughly in water. The most useful indicator of a toxic effect was the ratio of the rates of lung and buccal ventilation (which we term the 'R value'), which increased in affected frogs. The ventilation patterns of Rana pipiens, a species morphologically similar to the common frog, are described in detail by West and Jones (West and Jones, 1975). Early experiments (Oldham et al., 1993) showed that when the R value reached unity, that is when each breath results in lung inflation, a toxic effect was indicated and continued exposure resulted in death. All our work was conducted under licence from the UK Home Office and a condition of the licence was that distress and mortality of frogs should be minimised. Accordingly experiments were terminated, and the frogs washed thoroughly, whenever the R value approached unity. This often happened suddenly and in practice frogs were observed closely if the R value rose above about 0.4. High R values were not a characteristic of control frogs, in which any decrease in buccal venti-

R.S. Oldham et al./ Agriculture, Ecosystems and Environment 61 (1997) 69-74

lation was normally accompanied by a corresponding decrease in lung ventilation; R rarely rose above 0.4. From a practical standpoint, in order to minimise distress to the animals, an EC50 was calculated using Weil's method (Weil, 1952) which can provide statistically significant results using as few as three test animals per treatment. An R value approaching unity was taken as the end point, at which a toxic effect was indicated. As a demonstration that the R value is quantitatively related to the level of exposure to fertiliser, an analysis of variance was conducted on

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Time (mins.) Fig. 1. (a) Ventilation rates and R values (ratio of lung to buccal ventilation rates) of a control frog held in a laboratory aquarium on damp filter paper at 20°C. (b) Ventilation rates and R values of a frog held in the laboratory on damp filter paper with 12.4 g m - 2 of ammonium nitrate granules at 20°C. (c) Ventilation rates and R values of a frog held in an enclosure in a field of rye grass immediately after the application of ammonium nitrate granules at a concentration of 19.9 g m - 2 , at 17°C.

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R.S. Oldham et al. / Agriculture, Ecosystems and Environment 61 (1997) 69-74

dom, after ammonium nitrate had been applied using a 'waggling spout' apparatus at rates of 10.8 g m -2 on the wheat field and 19.9 g m -2 on the grass. Areas to be used in the control trials were covered with plastic sheeting during fertiliser application. One male frog was introduced into each of the three treated enclosures immediately after the fertiliser had been applied and one more into each of three identical enclosures on untreated plots. The frogs were regularly monitored for symptoms of toxicity over a period of 1 - 2 h, using the criteria described above. All were removed and washed as soon as symptoms of toxicity were shown. The mean temperature during the experiments ranged between 14° and 17°C. 2.3. Persistence o f toxicity

To examine the persistence of the acute toxic effect of ammonium nitrate granules, frogs were exposed in the laboratory, in two sets of trials, to soil which had been treated with fertiliser granules for different lengths of time (intervals of 1 h or 30 min) prior to their introduction. In the first set of trials, 14 identical experimental units were each provided with 2 cm depth of moist soil (22% water) and half of them treated with ammonium nitrate granules at field concentration (49.4 g m - 2 ) . The other half were used as controls. A male frog was introduced into one of the treated units immediately after fertiliser distribution, and into one of the control units at the same time. Each was observed as in the previous experiments. After 1 h frogs were introduced into two more units (treated and control) and again observed. The same pattern was repeated at hourly intervals until all the units had been used. In the second set of trials the same procedure was followed, but using only eight units, drier soil (7% water) and frogs introduced at 30 min intervals.

3. R e s u l t s

3.1. Recognition o f toxic symptoms

Characteristic patterns of ventilation rates are shown in Fig. 1. Fig. l(a), for a control frog, illustrates the erratic nature of the ventilation seen after introduction to the aquarium. The rates gradually

subsided and then remained stable for the 6 h of exposure. Fig. l(b) illustrates the ventilation rates of a frog exposed to ammonium nitrate granules, in the laboratory, on a paper substrate, at a concentration of 12.4 g m -2. The convergence of the two ventilation rates, representing an increase in the R value, was apparent after only 15 min and R reached unity after 30 min. At lower fertiliser concentrations and on uneven surfaces the increase in the R value was more gradual. On soil an effect was only evident after 80 min at a concentration of 12.4 g m -2, presumably because of a lower level of contact between fertiliser and skin. There was a similar trend with field exposure. Fig. l(c) illustrates the effects of exposure on grass in spring (19.2 g m - 2 , 17oC). An effect is evident, but at a higher concentration than would have been expected in the laboratory on a paper substrate. 3.2. Laboratory trials on acute toxicity

Results of the laboratory trials are summarised in Table 1. Frogs exhibiting symptoms of acute toxicity, which we predicted would lead to death, were removed from the experimental unit at the times indicated; the mean terminal R value was 0.7. All of these frogs recovered except two; the frogs removed from the paper and soil substrates (6.2 g m -2 and 12.4 g m -2 concentrations, respectively) after 180 rain and 15 min, respectively, appeared to recover normally, but subsequently died. The EC50 (the con-

Table 1 Acute toxicity of granular ammonium nitrate to adult male common frogs - - summary of laboratory trials Fertiliser concentration

Substrate F r o g s Frogs Time of tested affected effect(min)

(g m -2 )

0 1.5 3.1 6.2 12.4 0 3.1 6.2 12.4 24.8

Paper Paper Paper Paper Paper Damp soil Damp soil Damp soil Damp soil Damp soil

12 3 3 3 3 12 3 3 3 3

0 0 2 2 3 0 0 1 3 3

180, 300 180, 300 5, 15, 120 60 15, 90, 360 10, 10, 15

R.S. Oldham et al./ Agriculture, Ecosystems and Environment 61 (1997) 69-74 Table 2 Acute toxicity of granular ammonium nitrate to adult male common frogs - - summary of field trials Fertiliser concentration (gin -2 )

Crop

Frogs tested

Frogs affected

0 10.8 0 19.9

Wheat Wheat Grass Grass

3 3 3 3

0 3 0 3

73

5-110

concentrations (0-12.4 g m -z, Table 1) on a paper substrate, where n =- 3 in each case, was statistically significant (F4,t0 = 7.91, P < 0.05), high R values being associated with high concentrations of fertiliser. The regression of the mean R value against ammonium nitrate concentration showed a highly significant linear relationship ( t 3 = 2 0 . 0 , P < 0.001).

24-50

3.3. Field trials on acute toxicity

Time of effect (min)

centration producing an effect in 50% of the treated animals) was calculated using Weil's method (Weil, 1952). On a paper substrate the ECs0 was 3.6 g m -2, with a 95% confidence interval of (11.0, 1.2) g m -2. On a soil substrate the ECs0 was 6.9 g m -2 (95% confidence intervals of 15.5, 3.1 g m-2). Upper confidence limits on both substrates lie within the range of field nitrate applications used in the UK. The relationship between the R value and ammonium nitrate concentration was investigated using a one-way analysis of variance (after stabilising the variance of R by arcsine transformation). In each of the experiments R was calculated at 5 min intervals during the first hour (or until an effect was evident; Section 2.1). The difference in the mean value of R, during this period, at five separate ammonium nitrate

In both field trials all the frogs exposed to ammonium nitrate granules displayed symptoms of toxicity after 5 - I 10 min from the start of exposure (Table 2) by which time the granules had almost completely dissolved. Following removal all except one, from the grass environment, recovered. 3.4. Persistence of toxicity The fertiliser granules were visible on the soil surface for about 1 h in the first set of trials and for about 2 h in the second set. The toxic effect of the granules appeared to last longer on drier soil (Table 3). Application of Weil's (Weil, 1952) analysis to the data in Table 3 shows that a toxic effect occurred in 50% of frogs introduced to the tank when the fertiliser had been on dry soil (7% moisture) for 107 min, but on moist soil (22% moisture) for only 84 min. There was no evidence of a toxic effect once

Table 3 Persistence of toxic effect of granular ammonium nitrate applied at a concentration of 49.4 g m - 2 , to male common frogs Time between fertiliser application and frog introduction

Mean soil moisture content (%)

Mean temperature (°C)

No. of frogs tested

No. of frogs affected

Time of effect (min)

No. of control frogs tested

No. of control frogs affected

0 1h 2h 3h 4h 5h 6h 30 min 60 min 120 min 240 rain

22 22 22 22 22 22 22 7 7 7 7

20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.3 20.3 20.3 20.3

2 2 2 2 2 2 2 3 3 3 3

2 2 0 0 0 0 0 3 3 1 0

5, 5 25, 60

2 2 2 2 2 2 2 3 3 3 3

0 0 0 0 0 0 0 0 0 0 0

5, 5, 40 5, 5, 5 5

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R.S. Oldham et a l . / Agriculture, Ecosystems and Environment 61 (1997) 69-74

the granules had dissolved, even at the high application rate used.

4. Discussion These results provide the first evidence that a toxic effect occurs following direct contact between the frog and ammonium nitrate under laboratory and field conditions at rates comparable to those in the field. This toxic effect usually leads to death. However, no immediate effect is apparent once the granules have dissolved. The rapid dissolution of the granules under normal field conditions implies that frogs in treated fields will be exposed for a limited period and an acute effect would be expected for only an hour or so after application. Since fertiliser application is likely to be during daylight and most amphibian migration occurs during the hours of darkness there is a low probability o f an acute toxic impact o f ammonium nitrate on adult frogs under normal field conditions. The results presented here reveal unexpected acute toxic and lethal effects from a widely used material. In view of this, further work is necessary to demonstrate whether fertiliser might have other effects on frog populations in the field. There remain at least four avenues through which fertilisers might affect frog populations: 1. through more slowly dissolving fertilisers, such as muriate of potash and ' G r o w m o r e ' ; 2. through acute effects on eggs and larvae (Berger, 1989) or on adult frogs as a result of fertilisers leaching or runoff into aquatic breeding sites; 3. through acute effects on juveniles, and perhaps females, which may be more sensitive than adult males; 4. through sublethal impact, frogs escaping mortality

but suffering reduced growth or reproductive output.

Acknowledgements The work was supported by funding from the Nature Conservancy Council and English Nature. W e thank N. Renner for permission to work on his land, C. Draper for information on fertilisers, J. Brooks, P. Guirmess, S. Hill and J. Johnson for laboratory assistance and D.J. Bullock and J.A. Fowler for criticism of the manuscript.

References Agricultural Development and Advisory Service, 1992. Survey of Fertiliser Practice: Fertiliser Use on Farm Crops in England and Wales 1992. Agricultural Development and Advisory Service, Edinburgh. Berger, L., 1989. Disappearance of amphibian larvae in the agricultural landscape. Ecol. Int. Bull., 17: 65-73. Chalmers, A., Kershaw, C. and Leech, P., 1990. Outlook Agric., 19: 269-278. Cooke, A.S., 1972. Indications of recent changes in status in the British Isles of the frog (Rana temporaria) and the toad (Bufo bufo). J. Zool. Lond., 167: 161-178. Cooke, A.S. and Scorgie, H.R.A., 1983. Focus on Nature Conservation. Nature Conservancy Council, Peterborough. Hilton-Brown, D. and Oldham, R.S., 1991. The status of the widespread amphibians and reptiles in Britain, 1990, and changes during the 1980's. Nature Conservancy Council Contract Survey, Nature Conservancy Council, Peterborough. Oldham, R.S., Latham, D.M., Hilton-Brown, D. and Brooks, J.G., 1993. The effect of agricultural fertilisers on amphibians. English Nature Contract Report, English Nature, Peterborough. Weil, C.S., 1952. Tables for convenient calculation of median-effective dose (LDso or EDso) and instructions in their use. Biometrics, 8: 249-263. West, N.H. and Jones, D.R., 1975. Breathing movements in the frog Rana pipiens. 1. Mechanical events associated with lung and buccal ventilation. Can. J. Zool., 53: 332-344.