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Effects of Pesticides on Bombina bombina in Natural Pond Ecosystems

6 Discussion

6.1 Pesticide concentrations

There are very few reports on pesticide contamination of ponds. On the contrary, pesticides in ditches, streams and watersheds have been examined numerous times. Therefore concentrations of pesticides in running waters has to be included for evaluation of results obtained in this investigation.

6.1.1 Herbicides

Concentrations exceed previous Danish results

Herbicides ranged up to 11.44 μg/l in this investigation and were detected in ponds P1-P8 and P10. The concentrations exceed previous results from four Danish ponds where mechlorprop, 2,4-D and MCPA were found in concentrations between 0.1 and 1.1 μg/l (Mogensen & Spliid 1997). In a southern Danish stream on clay soils, Højvands rende, mechlorprop and other phenoxy acids were found in concentrations of 20-5300 ng/l (Mogensen & Spliid 1997). In a stream in Fyn County situated in an agricultural landscape maximum concentrations of the herbicides bentazone, hexazinone, isoproturon and mechlorprop from 1994-96 were between 1 and 10 μg/l (Wiberg-Larsen & Pedersen 1997). Out of the totally 19 detected pesticides in the present investigation 14 were herbicides. Herbicides generally seemed to be more persistent in the pond water than the insecticides and fungicides and often two or more herbicides were present in the same sample. In accordance with this Spliid (2001) found herbicides more frequent than insecticides and fungicides in drainage water in Danish clay soils. In southern Sweden Kreuger (1998) found that the clear majority of pesticides found in stream water in an agricultural catchment area were herbicides and some of the herbicides were found to persist during a year after spraying.

6.1.2 Insecticides

Normal concentration in Denmark

Insecticides were found in concentrations up to 460 ng/l (pirimicarb) which is in accordance with concentrations found in surface waters in other investigations in Denmark. In Ringkøbing County, dimethoate and pirimicarb were found at concentrations of 560 ng/l and 230 ng/l respectively (Mogensen & Spliid 1997). In Fyn County, dimethoate and pirimicarb have reached concentrations of 200 ng/l and 600 ng/l respectively (Wiberg-Larsen & Pedersen 1997). Peak surface water concentrations of 2.5 mg/l of the pyrethroid insecticide fenitrothion in small Canadian forest ponds have been reported by Ernst et al (1991). The high level of insecticide was reached as a result of spraying by aircraft and the samples were taken 15 minutes after the spraying was performed. Mean surface water concentrations ranged from 0.04 to 1.5 mg/l in six ponds. In Kansas, dimethoate reached 4.3 μg/l at maximum and a mean of 1.9 μg/l in tailwater drainage pits collecting run-off irrigation water (Kadoum & Mock 1978).

Insecticides were generally found in much lower concentrations than herbicides and the concentration of insecticides diminished quickly after application. Pirimicarb was reduced from 240 to 60 ng/l from the day of spraying till 2 days after. In tailwater drainage pits, Kadoum & Mock (1978) found herbicides to occur more frequently than insecticides, and insecticidal residues were usually not found at the end of the growing season. Heinis & Knuth (1992) showed that 90 % of the applied amount of esfenvalerate disappeared from the water column within the first 24 hours after spraying with an initial concentration of 5 μg/l. Mogensen et al (in press) have found fast disappearance of the pyrethroid insecticides fenpropathrin, permethrin, esfenvalerate and deltamethrin from the water column in artificial experimental ponds. The insecticides were sprayed onto the surface of the ponds and were evenly distributed in the water column within the first day. The concentration in the water decreased fast mainly due to adsorption of the insecticides to sediment particles.

Low persistence of insecticides

Low persistence of insecticides

Pyrethroid insecticides adsorb fast to vegetation, algae, suspended organic matter and sediment and thereby complicate detection and evaluation of risk (Heinis & Knuth 1992; Pedersen 1999). Insecticides are designed for rapid disappearance from the environment and are often highly degradable (Smith & Stratton 1986). Rapid degradation and adsorption may explain the frequent observations of no insecticides or only low concentrations in the ponds following application to surrounding fields.

Possibly higher peak concentrations

Out of seven detections of pirimicarb in 1995, the highest concentration was 460 ng/l on the 9th of July. The sample was taken a few hours after application in a pond with a 10 m wide buffer zone. It is likely that other ponds with more narrow buffer zones might have reached higher initial concentrations of insecticides than the highest detected values of 130-150 ng/l. If samples in all cases had been taken within a few hours after spraying, the actual maximum concentrations would have been obtained. Such a procedure was impossible in this investigation.

6.1.3 Fungicides

Generally low persistence

Like the insecticides, the fungicides fenpropimorph and propiconazol were less persistent than the herbicides. In P8 in 1994 the persistence of fenpropimorph and propiconazol could be compared. Although it is prohibited to use fenpropimorph and propiconazol within 20 m from lakes (Petersen et al 1998), the water in pond P8 was contaminated with both fungicides three times in the spring 1994, possibly as a result of spray drift. In all three cases the concentration of fenpropimorph decreased much faster than the concentration of propiconazol. In a pond water/sediment system the degradation time (DT50) to half concentration was 5 days for fenpropimorph compared to 30 days for propiconazol (DEPA 1996a, DEPA 1996b). Given the low persistence of especially fenpropimorph it is obvious that some of the peak concentrations reported easily could have been much higher.

6.2 Sources of contamination

Spray drift

Rain

Spray drift of pesticides was presumably the major source of contamination of ponds. However other sources may also have been involved. In the present investigation phenoxy acids were often found in concentrations of 10-350 ng/l, even though the same herbicides were not applied to the surrounding fields that year. Contamination of pond water by rain is a possibility as rain water in Denmark can contain up to 400 ng/l of MCPA and dichlorprop, and up to 130 ng/l of mechlorprop (Mogensen & Spliid 1997).

Phenoxy acids can also appear in high concentrations in groundwater in Denmark (Mogensen & Spliid 1997). Many herbicides are persistent in the environment and may wash out from the top soil to surface waters either by run-off incidents, or through horizontal migration of pore water during heavy rain fall or by drainage water (Wauchope 1978; Kadoum & Mock 1978; Nicholaichuk & Grover 1983; Thurman et al 1992; Klöppel et al 1994; Aderhold & Nordmeyer 1995). In Danish sand and clay soils sudden leaching of several pesticides to drainage depth as a result of short rain events has been shown with concentrations up to 2.84 mg/l (Spliid & Køppen 1998; Spliid 2001). In P10, which is situated in a camping site on the east coast of Fyn, 580 ng/l of 2,6-dichlorbenzamid also known as BAM was found in December 1997. BAM is a breakdown product of the herbicide dichlobenil. Dichlobenil has probably been used to remove vegetation from small unpaved roads on the camping site. The pond P10 is situated about 30 m from an unpaved road. After breakdown of dichlobenil BAM has probably been washed into the nearby pond through the soil.

Buffer zone can minimize transport

Run-off

An established vegetation in a buffer zone can filter out eroded soil during run-off incidents and thereby minimize the transport of pesticides adsorbed to soil particles (Nicholaichuk & Grover 1983; Klöppel et al 1994). All ponds were filled by surface water and water originating from secondary ground water sources. P5 also had an inlet of drainage water from 2-3 hectares of farmed land. Some contamination of pesticides has undoubtedly been transported to the ponds by run-off surface water, migration of pore water or by drainage water in P5. It was not possible to detect any influence of the drainage water on the contamination of pond P5 with pesticides. Pond P5 was one of the least contaminated ponds.

6.3 Buffer zone and spray drift

Buffer zones reduced contaminations

The buffer zones were found to reduce contamination of the ponds with pesticides. Findings of pesticides in ponds was reduced significantly by increasing width of buffer zone between 1-10 m. There was also found a tendency (non significant) towards decreasing maximum concentrations of pesticides with increasing width of the buffer zone (1 to 10 m). Maximum pesticide concentrations during the three year investigation period never exceeded 0.5 μg/l in ponds with buffer zones of more than 4 m but was above 1 μg/l in ponds with buffer zones of 4 m or less in 4 out of five cases. The results show that a buffer zone reduces the deposition of pesticides in ponds probably by filtering out pesticides in the vegetation. But even a buffer zone of 10 m did not offer complete protection against contamination with pesticides.

6.3.1 Establishing a safe distance

Safe distance varies

between organism and lifestyle

Establishing safe dimensions for buffer zones is a vital issue and a matter of increasing concern in Denmark. The width needed depends on the organism or life stage that is in focus. According to de Snoo & de Wit (1998) a buffer zone of only 3 m decreases drift deposition in a ditch by 95 % and a buffer zone of 6 m eliminates drift deposition. The results from de Snoo & de Wit was found using water sensitive paper and it is doubtful if the safe distance given is adequate in all circumstances given the results in the present investigation. Other investigators have shown that at least for some species, younger plants are more susceptible to herbicide drift than established plants (Marrs et al 1991; Marrs et al 1993). This may alter the ecological balance by reducing the diversity of plant species in field margins and buffer zones (Marshall 1987), or by replacing sensitive species by resistant species (Marrs & Frost 1997). In earlier experiments with established perennials, the width of buffer zones sufficient for protection of vegetation were estimated to be 6-10 m (Marrs et al 1989). Later experiments with seedlings have shown that buffer zones must be 20 m wide to protect regeneration of vegetation by seedlings (Marrs et al 1993). Extremely sensitive plant species may need a safe distance of 100 m or more for complete protection (Nordby & Skuterud 1975; Byass & Lake 1977). Davis et al (1993) showed in an experiment with the highly toxic pyrethroid insecticide cypermethrin that safe distances for reducing mortality of butterfly larvae (Pieris brassicae) to 10 % were between 16 to 24 m.

Buffer zones of 10 m are not enough

In conclusion, most results comply with findings in this investigation that buffer zones of 10 m are not sufficient to protect sensitive environments from spray drift of pesticides.

6.3.2 Height of vegetation

No effect of height of vegetation

The height, structure and density of the vegetation in buffer zones will affect the turbulence of a drifting cloud of pesticides and thereby alter the probability of deposition within the buffer zone (Marrs et al 1993). By increasing vegetation height, the roughness coefficient, Z0, and the eddy or frictional velocity, U*, increases, causing increased deposition of small droplets. How ever in this investigation the height of the vegetation in the buffer zone (0.1 - 1.0 m) did not correlate with maximum pesticide concentrations in ponds.

Deposition of spray complex

Greater risk in spring and autumn

It seems surprising that buffer zone height had no effect on maximum pesticide concentrations in this investigation. The reason may be that the number of investigated ponds or the span of height of buffer zone (0.1 - 1 m) were too small to yield a statistical difference. The lack of comparability in methodology, e.g. in density and structure of vegetation, boom height, working pressure, wind speed and time elapsed between spraying and sampling, makes the comparison of results and detection of differences difficult. Marrs et al (1991) did not either find a connection between height of vegetation in buffer zone and spray drift, but ascribed the missing connection to the complex pattern of deposition of spray under field conditions. Davis et al (1993) failed to connect deposition of drifting droplets to vegetation height due to large variations in methodological variables. Longley & Sotherton (1997) found that autumn spraying, when compared to summer spraying, involved greater risks of spray drift because of lower vegetation in both field and buffer zone. Spraying in spring compared to summer should therefore also involve greater risks.

Hedges limits spray drift

To effectively reduce a drifting cloud of pesticide within a short distance the vegetation of the buffer zone has to be significantly higher than the crop. Hedges in the buffer zone limits spray drift significantly (Davis et al 1993; Longley et al 1997). Planting a hedge consisting of 2-3 m high bushes in the buffer zone should increase the ability of the buffer zone to filter out pesticide drift, thereby preventing contamination of ponds. Amphibians breed in partly or fully sun-exposed ponds and therefore it is important not to plant bushes or trees that will shade ponds.

6.3.3 Practical measures to minimize spray drift

Wind speed

Boom height and working pressure

A few measures can be taken to effectively minimize spray drift. Maybank et al (1978) found that the amount of drift increased linearly with the wind speed (2–7 m/s). Spraying should therefore be carried out in the lightest wind possible (Maybank et al 1978) and not stronger than 3 m/s (Nordby & Skuterud 1975; Longley & Sotherton 1997). Wind speeds often vary during

the day and are normally lower in early morning than later in the day. Spraying in the morning is therefore recommended. The boom height and working pressure has a huge effect on drift (Nordby & Skuterud 1975; Maybank et al 1978). Spraying with a boom height of 40 cm instead of 80 cm and a working pressure of 2.5 bar instead of 10 bar, reduced initial drift by 50 % (Nordby & Skuterud 1975). Spray drift will be reduced if spray droplets are changed from fine to coarse (Murphy et al 2000). Maybank et al (1978) also point out that the use of thickeners and application of higher total volume can reduce the level of initial drift.

6.3.4 Effect of farm size

In 1993, high concentrations of pesticides (up to 7.475 μg/l) appeared in ponds on the relatively small farms/fields on Avernakø (P1-P7), and low concentrations (<130 ng/l) appeared in pond P8 on the large farm/field on the east coast of Fyn. In 1994 the maximum concentrations were a few hundred ng/l on Avernakø (P1-P7, P9), but more than 11 g/l in P8 on the large farm. In 1995 again there were peak concentrations of up to 3 μg/l in P8, whereas concentrations on Avernakø (P1-P7, P9) did not exceed 500 ng/l. Observations of tracks in the field made during spraying showed that spraying was done at distances from the ponds that varied from year to year. This may be the main reason for the large year-to-year variation. Therefore this investigation cannot conclude that a certain size or type of farm causes less chance of contaminating ponds with pesticides. Cautiousness from the workers is probably far more important.

6.4 Food Quality Index

High FQI increased growth

Bombina bombina tadpoles feed upon epiphytic layers of algae, bacteria, protozoa, ciliates and rotifers on stems of plants (Mossin 1988; Andersen 1992). Established plant communities were found in all ponds. Cages and bags served as substrates for epiphytic growth in the experiments, and thereby acted as a substitute for the lack of vegetation in the enclosures. An increase in FQI, the amount and quality of food available to tadpoles, had a positive effect on the growth of the tadpoles.

Effect of herbicides in P8

No direct effect on algae

There were no significant correlations between FQI and use of herbicides, fungicides or insecticides. Since algae diversity is the main component of FQI, it was expected that herbicides could have an influence on FQI. Only in P8 a connection between herbicide contamination and FQI was revealed. The amount and diversity of food sources for the tadpoles were very low in P8 in 1995 due to a very low diversity of algae and due to the absence of Leptothrix bacteria. Maximum concentrations of herbicides were 11.4 μg/l of mechlorprop in 1994, and 2.1 μg/l of clopyralide, and 2.3 μg/l of propyzamide in 1995. The high level of herbicides was possibly the main reason for the complete dying out of the dominating Sparganium erectum vegetation in the pond. The decay of a complete vegetation releases a large amount of nutrients and the ecosystem was altered dramatically when the vegetation changed from macrophytes to a dense cover of filamentous algae, keeping out all light and thereby reducing the biomass and diversity of epiphytic algae.

Probably no direct toxic effects of herbicides on algae occurred in P8 but the effect of the herbicides were indirect by removing the plants which the epiphytic community of algae, bacteria and protozoa depends up on.

Hormone type herbicides

Hormone type herbicides like MCPA and 2,4-D have EC50 values of 85 and 98 mg/l on a Scenedesmus algae (Fargasova 1994). In another investigation, the application of 2,4-D, resulting in a concentration of 140 g/l, eliminated all plants in an irrigation channel (Akinyemiju & Bewaji 1990). In this investigation residues of 2,4-D were found in most of the ponds, but the concentrations were below detection limits (10-500 ng/l). MCPA reached a maximum concentration of 172 ng/l in P2 in 1993. For hexazinone the EC50 for growth inhibition of green algae, diatoms, duckweed and cyanobacteria were respectively 10, 50, 70 and 600 g/l (Peterson et al 1997). In this investigation hexazinone was only detected in P7 in 1995 at a concentration of 70 ng/l. Hormone type herbicides did probably not occur in harmful concentrations in the ponds in this investigation.

Effect of propiconazol

In P8 concentrations of propiconazol reached 1.214 μg/l and 2.968 μg/l in 1994 after spraying of the formulated product Tilt Megaturbo (active ingredient fenpropimorph and propiconazol) 3 times (April, May and June). According to DEPA (1996b) the EC50 of propiconazol is 9 mg/l for the aquatic plant Lemna gibba (duckweed), 93 μg/l for the algae Navicula seminulum and 21 μg/l for the marine algae Skeletonema costatum. When using the formulated product Tilt 250 EC (active ingredient propiconazol) effects on algae appear at much lower concentrations. In a five days experiment with Tilt 250 EC half of the algae Skeletonema costatum was killed by 0.7 μg/l of propiconazol (DEPA 1996b).

No direct effect on algae

It seems obvious that in this investigation the contamination of herbicides was too small to have a direct effect on algae diversity. But the contamination of P8 in 1994 with the fungicide propiconazol may have affected the algae. The Food Quality Index is unfortunately not available from P8 in 1994 and it is not known what the initial concentrations were in the pond just after the pesticide spraying. Further, there are also too few reports available concerning toxicity of formulated pesticides towards fresh water algae. Therefore no conclusions can be drawn.

Effect on protozoa and other small organisms

The herbicide isoproturon has a negative effect on the ciliate Tetrahymena with EC50 of >1.1 mg/l (Traunspurger et al 1996). In the same investigation no effects of isoproturon were observed on rotifers. The effects of three insecticides on Tetrahymena were tested in the laboratory (Larsen 1997). There were no effects of cypermethrin at 20 μg/l, which was the highest concentration tested. Pirimicarb and dimethoate showed a dose dependent effect on growth at concentrations above 200 and 500 mg/l respectively. A concentration of 50 μg/l of the pyrethroide permethrin kills rotifers effectively in experimental lake enclosures. The effect is short-lived however, and the population can recover within 3-4 weeks (Smith & Stratton 1986). Only at concentrations exceeding 50 mg/l there has been demonstrated an effect of propiconazol on protozoa (DEPA 1996b).

It seems very unlikely that pesticides had a direct effect on protozoa and other small animals in this investigation.

Leptothrix bacteria are known to be an important food source for Bombina bombina tadpoles in some ponds (Mossin 1988). Very few data are available on the effects of pesticides on microbes. Smith & Stratton (1986) report that the insecticide permethrin, when tested in situ at concentrations up to 10 mg/kg in mineral and organic soils, showed no long-term effect towards numbers of bacteria and fungi. The methane production of a freshwater sediment was almost completely inhibited as a result of 90 μg/l of isoproturon (Traunsburger et al 1996). Probably the inhibition of methanogenesis was caused by one or more metabolites of isoproturon degradation. Another herbicide, simazine reduced methanogenesis in homogenised sediment samples at concentrations of 20 mg simazine/l when incubated for one week (Isolda & Hayasaka 1991).

Due to the very high, effective concentrations of pesticides reported and low concentrations obtained in this investigation, it is not likely that pesticides had an effect on the diversity and quality of food for the tadpoles except indirectly in P8.

6.5 Survival and growth of eggs and tadpoles

6.5.1 Hatching of eggs

Hatching in spraying season

Except for P3 in 1995 hatching of eggs was almost 100 % successful in all ponds, which reflects that the eggs were of a high quality. Hatching of eggs occurred during the end of May and beginning of June which coincided with the middle of the spraying season. In May 1995 the herbicides tribenurone and fluroxypyr were sprayed around P3 and 4 days later it was observed that the development of about half of the embryos was arrested. Only 47 % of the eggs hatched. Tribenurone and fluroxypur could not be detected by the laboratories and contamination of the pond could therefore not be verified. The pyrethroide insecticide fenitrothion (8 mg/l) and the herbicides triclopyr (4.8 mg/l) and hexazinone (100 mg/l) had no effect on hatching of embryos of Rana pipiens, Rana clamitans and Rana catesbeiana under laboratory conditions (Berrill et al 1994). According to Larsen & Sørensen (2004), periodic spasmodic twisting was induced in embryos of both Xenopus laevis and Bombina bombina at 1 μg/l of the pyrethroide esfenvalerate (LOEC, 120 hr). Malformations of embryos of B. bombina increased with increasing concentration of esfenvalerate from 5 μg/l (LOEC) with an EC50 value of 29 g/l. No inhibition of growth or increased mortality of embryos was found at the highest tested concentration, 150 μg/l. No levels of pyrethroids in this investigation even came close to the LOEC of esfenvalerate found by Larsen & Sørensen (2004). In conclusion, the embryo when still inside the egg was probably not a particularly vulnerable life stage to pesticides for B. bombina in this investigation.

6.5.2 Survival of tadpoles

Small tadpoles vulnerable

The survival of the tadpoles was often negatively affected during the first 14 days after hatching indicating that this life stage is one of the most vulnerable periods in the life of tadpoles. Berrill et al (1997) have found that newly hatched tadpoles are vulnerable to exposure to low concentrations of herbicides and insecticides. Bromoxynil caused extensive mortality among Rana clamitans tadpoles at 10 μg/l, and the pyrethroids permethrin and fenvalerate caused paralysis or at least much weakened responses in tadpoles of R. clamitans and Rana pipiens also at 10 μg/l. The embryos of the same species were unaffected by exposure to the same concentrations of pesticides (Berrill et al 1997). This fact underlines the vulnerability of the newly hatched tadpoles.

Predators important

There seems to be a number of possible threats to tadpoles besides the pesticides. In 1993-94 the experimental bags were made of large-meshed nylon (1*1 mm) which made entering of small leaches possible. Intruding leaches were found to have caused the death of all tadpoles in 2 cases from 1993-94. New bags of finely-meshed nylon (0.1*0.1 mm) were used in 1995 which made it possible to separate the tadpoles from predators. In 7 out of 10 cases of low survival from 1993-95, sulphur bacteria (indicating a low oxygen concentration) were abundant. There were no cases of good survival when sulphur bacteria was abundant. The young tadpoles do not breathe by lungs and therefore depend solely on the oxygen in the water. They also have a low mobility in this stage. It is therefore possible that they were killed either because of a low oxygen concentration or because of the presence of the highly toxic H2S.

Fungicide correlation coincidental

There was a significant negative correlation between fungicide concentration and survival of 14-30 days old tadpoles in 1995. But only minor concentrations of fungicides were found in the ponds this year. Given all the other possible mortality factors for newly hatched tadpoles mentioned above, the correlation might as well be coincidental.

Large tadpoles less vulnerable

The cage experiment started with tadpoles of 0.2 g. At this stage the tadpoles are very mobile and therefore have a good chance of escaping intruding predators or areas with low oxygen tension or presence of H2S. Furthermore, they have developed lungs and are able to breathe air at the surface. Tadpoles in ponds with large coverings of sulphur bacteria and large numbers of predators such as leaches, did not have the same low survival rate as demonstrated for the small tadpoles in the bag experiments.

Small variations in water temperature

Survival of tadpoles to metamorphosis was negatively correlated with herbicide concentration and positively correlated with FQI in 1995. It could be that the herbicides primarily reduced FQI and thereby indirectly reduced the fitness of the tadpoles. But these correlations might also be coincidental. In 1995 no significant correlations could be found between herbicides and any other growth parameters.

Herbicide contamination – low survival of tadpoles

The herbicide contamination was worst in P8 in 1995, when the lowest survival of tadpoles occurred with only one out of 40 surviving to metamorphosis. In 1993 and 1994 the highest survival of the large tadpoles in cages was found in P8. It is not likely that the tadpoles were killed by the herbicides in 1995. According to Cooke (1972), the herbicide 2,4-D had no effect on tadpoles of Rana temporaria and Bufo bufo in a laboratory experiment even at 50 mg/l. In a laboratory bioassay Sanders (1970) showed that 2,4-D has an LC50 value of 100 mg/l towards Pseudacris triseriata tadpoles. The reason for the very low survival may be that the ecosystem in the pond was completely altered after the disappearance of the dominating macrophytes. Although all filamentous algae were removed from the cages every week the layer of algae became so dense between the weekly cleanings that the tadpoles might have been caught in it and killed during the night due to depletion of oxygen. The food sources of the tadpoles disappeared almost completely as the algae diversity was reduced to a thin layer of only two species in May. Also the low water level in the end of the experiment (10 cm) in combination with a probably high oxygen demand of the decomposing vegetation have probably stressed the tadpoles. Thick coverings of sulphur bacteria were found in the cages in P8 in 1995. Poor conditions for the tadpoles could have made them more susceptible to predation by leaches that could enter the cages. The growth rate of the tadpoles was at average in 1995 although the survival was very low and therefore not as poor as could have been expected due to the very low FQI. A possible explanation might be that the tadpoles utilised epiphytic coverings in areas periodically open due to the weekly removal of filamentous algae. By the decay of the macrophytes a large amount of dissolved organic material was probably released to the water. The tadpoles might also partly have lived on filtering out the dissolved organic material from the water as the epiphytic food sources were very scarce.

6.5.3 Growth of tadpoles

From a previous investigation it is known that the water temperature has some importance for growth of Bombina bombina tadpoles (Andersen 1992). In this investigation however, there were no correlations between water temperature and parameters regarding hatching of eggs, growth of tadpoles or weight and time at metamorphosis. The reason is probably that all ponds were selected to be shallow and fully exposed to the sun in order to minimize differences in temperature between ponds. Also an effect of differences in temperature between ponds was partly counterbalanced since the tadpoles would search the optimal temperature in the cages. There are nevertheless some indications from this investigation that temperature could be of importance during the weeks of the cage experiment. The lowest growth rate for tadpoles in week one was found in P5 and in P6 in 1994 where also the lowest water temperatures in the experiment were recorded. In 1995 the lowest growth rate of tadpoles in week one and the lowest temperature was found in P5 and P7. Besides these examples, no other effect of temperature on growth rate could be found.

6.5.4 Metamorphosis

Small metamorphs in new ponds

The abundance of protozoa in P9 in 1994 may be the reason that the highest average metamorphosis weight during the investigation occurred in P9 that year. In P10 and P11 situated in a non-cultivated landscape the metamorphosis weight were low compared to the other ponds. The reason is probably that P10 and P11 were newly dug with a clay bottom and therefore presumably less eutrophic than the other ponds. Bombina bombina prefers natural eutrophic ponds with a thick sediment of mud (Andrèn et al 1984) which partly can be explained by a higher density of epiphytic growth on plant stems.

Effect of low water level

Only in P8 the water level decreased considerably in the cages during the experiment. In 1993 the tadpoles metamorphosed early in P8 compared to the other ponds. This may have been caused by the low water level, but in 1994 and 1995 no effect of low water level was seen on metamorphosis time. Amphibians are seen to accelerate their development if endangered by desiccation (Duellman & Trueb 1994).

Correlations with pesticides

In 1993 there were significant negative correlations between both fungicide and insecticide contamination and weight at metamorphosis. This was the only year where insecticides were sprayed on fields at five ponds and fungicides on fields at four ponds before mid June. Insecticides could be detected in three ponds and high concentrations of fungicides (3.550 μg/l and 7.475 μg/l) were found in two ponds this year. In 1994 insecticides were not detected, and in 1995 insecticides were sprayed in the period 25th of June to 9th of July. In 1995 weight at metamorphosis correlated significantly positive with fungicide and pesticide contamination. Much higher concentrations of fungicides were found in the ponds in 1993 than in 1995. Around four ponds the fields were sprayed with fungicides before mid June, and at three more ponds fungicides were sprayed on fields 25th of June in 1995. The potential contamination of ponds with fungicides was not less in 1995 than in 1993. Therefore the results are contradictory and offer no basis for conclusions. Only the negative correlations between both insecticides and fungicides and metamorphosis weight in 1993 make sense. But given the small scale of the investigation and frequency of contaminations of the ponds, the correlations can be coincidental.

Synergistic and additive effects

Synergistic (Lichtenstein et al 1973) or additive (Bailey et al 1997) effects are possible when pesticides appear together in the same environment. Synergism of organophosphorus, carbamate and chlorinated hydrocarbon insecticides by the herbicides atrazine, simazine, monurone and 2,4-D have been reported by Lichtenstein et al (1973). Fairchild et al (1994) failed to show synergism of the insecticide esfenvalerate by atrazine, probably because of rapid aqueous dissipation of esfenvalerate. Bailey et al (1997) have shown that the organophosphorus insecticides diazinone and chlorpyrifos exhibit additive toxicity. Synergistic or additive effects could not be shown in this investigation, but in P3, P4 and P6 in 1993, and in P8 in 1994 and 1995 high concentrations of fungicides and herbicides respectively were supplemented with the presence of other types of pesticides.

Effect of pesticides on tadpoles

Pesticides may influence the weight and fitness at metamorphosis by altering the behaviour of tadpoles or by inducing damages in tadpoles. Berrill et al (1993) found effects as delayed growth, abnormal backs and twisting behaviour in tadpoles of Rana clamitans from exposure of embryos to 0.1 mg/l of the pyrethroide permethrin. Periodic spasmodic twisting in embryos of Xenopus laevis and Bombina bombina was seen from 1 g/l of esfenvalerate (Larsen & Sørensen 2004).

Marian et al (1983) found that Rana tigrina tadpoles suffered significant reduced size at metamorphosis as a result of exposure to 0.1 mg/l of the insecticide carbaryl. Because pyrethroids are rapidly adsorbed to organic matter (Ohkawa et al 1980), tadpoles may increase their exposure to esfenvalerate by their feeding behaviour. Beginning malformations of the gut have been found in embryos of B. bombina at 5 μg/l of esfenvalerate by Larsen & Sørensen (2004). The effect was dose dependent and at 100 μg/l all embryos suffered from malformations in the gut, heart, abdomen and eyes. Xenopus laevis was also tested in this experiment and showed the same response to esfenvalerate but at much lower concentrations. Thus the EC50 for malformations of X. laevis and B. bombina was 3 μg/l and 29 μg/l respectively.

Pirimicarb and dimethoate are not as toxic as esfenvalerate but the concentrations needed to reduce activity in tadpoles are unknown. Pirimicarb has been shown to cause malformations of tadpoles of Rana perezi when exposed to concentrations of 250 μg/l (Alvarez et al 1995). Damages on vital internal organs and increased mortality of R. perezi tadpoles are caused by pirimicarb at concentrations of 70 mg/l (Honrubia et al 1993). Such high concentrations are not likely to have occurred in this investigation and therefore it is believed that insecticides did not have direct effects on the tadpoles of Bombina bombina.

In this investigation 3-4 different pesticides were often found in the same samples and some of the pond ecosystems were subjected to repeated exposures to pesticides at low concentrations. Reports on synergistic or additive effects on tadpoles by repeated exposures to low concentrations of the most commonly used fungicides and insecticides are not available at present. Such a investigation would provide a better basis for evaluation of the actual risks of amphibians under typical field conditions.

Size and fitness

The size at metamorphosis determines the size (males and females) and time (only females) of first reproduction in the newt Ambystoma talpoideum (Semlitsch et al 1988). A reduction in growth of tadpoles will increase risk of predation and delay the time of metamorphosis. Salamanders metamorphosing early were larger at first and second reproduction compared to late-metamorphosing individuals (Semlitsch et al 1988). A delay of metamorphosis also increases the risk of desiccation in temporal ponds which are preferred as breeding ponds by many amphibian species including Bombina bombina. Sublethal concentrations of pesticides can therefore reduce the reproductive potential of an amphibian population and thereby increase vulnerability to other threats. Induction of abnormal behaviour as partial paralysis and twisting will increase vulnerability to predation (Berrill et al 1993). Pesticide induced hyperactivity can also increase risk of predation. Cooke (1971) has shown that DDT-treated hyperactive tadpoles of Rana temporaria were predated selectively by the newt Triturus cristatus.

6.6 Survival of adults

Survival connected to degree of cultivation

The yearly survival of adults was in general higher in uncultivated areas than in cultivated areas. The yearly survival was up to 94 % in areas with less than 50 % of the habitat being cultivated. In areas with intensive cultivation as on Avernakø, the yearly survival did not exceed 60 %. Low adult survival (43 %) also occurred in one case in an uncultivated landscape (Knudshoved Odde) which could reflect natural oscillations in survival of an isolated population (Duellman & Trueb 1994). A 1990-cohorte of young Bombina bombina from the island Hjortø (50 % cultivation) had a significantly faster growth rate (1990-92) than a 1990-cohorte of young B. bombina from the island Avernakø (90 % cultivation) (Briggs 1993). The anuran species diversity and density in a vegetable growing area has been shown to be significantly lower relative to nearby uncultivated wetlands (Bishop et al 1999).

Mechanical disturbance

An increased mortality in agricultural habitats could be a result of mechanical disturbance by agricultural machinery. Dürr et al (1999) found that ploughing caused the death of more than 90 % of juvenile migrating amphibians because they were buried alive (including Bombina bombina). Cultivation of a stubble field did not damage amphibians, but mulching of plants with a flair mower caused the death of 30 % of Rana arvalis in wet spots in fields (Dürr et al 1999). Furthermore the lack of hideaways in a modern agricultural landscape increases the risk of desiccation and predation during migration. Especially for amphibian populations dependent on crossing large fields during migration ploughing and the lack of shelters are important factors for adult survival. On Avernakø, B. bombina often crosses fields to reach ponds or winter habitats several times during a year, provided the distance does not exceed 200-300 m (Briggs 1993). When migration coincides with cultivation of fields the death of a large percentage of the population of B. bombina is to be expected. Crops as winter wheat and winter rape requires ploughing of the fields in August-September which is coincident with the migration period of B. bombina. These crops are commonly used on Avernakø and this might partly explain the lower survival of B. bombina in this highly cultivated area.

Contact with fertilizer grains

Migrations across fields impose a serious risk to amphibians due to the possible contact with fertilizer grain and pesticides. Nitrate and potassium from the fertilizers easily diffuse across the highly permeable skin of amphibians and damage the nervous system (Nielsen & Schouboe 1990). Schneeweiss & Schneeweiss (1997) showed that fertilizers killed 75 % of spring migrating amphibians on a field the first three days after application of fertilizer. Repeating the experiment in the autumn resulted in the death of all migrating amphibians the first day after application. Both in spring and autumn there was zero mortality of migrating amphibians before application of fertilizer. Dürr et al (1999) found that fertilizer grains had almost no effect on migrating amphibians as long as the ground was not completely dry. The fertilizer grains take up water from the soil and this reduces the toxicity towards amphibians (Dürr et al 1999). On Avernakø fertilizer grains have been observed lying undissolved on the ground for 14 days from late April to early May. In dry and sunny periods B. bombina males are calling from the ponds, competing to attract females. Both sexes migrate from the hibernating sites to the ponds and between ponds during this period. According to Schneeweiss & Schneeweiss (1997) the frogs may suffer a high mortality during migration across fields with fertilizer grains.

Pesticides and survival

The knowledge concerning effects of pesticides on adult amphibians is limited. In field trials Dürr et al (1999) found no effects on juvenile Rana arvalis of spraying of the herbicide glyphosate on fields. DNA profiles can indicate whether a frog population is under pressure due to contamination with pesticides. Lowcock et al (1997) found a significant increase in abnormal DNA profiles in individuals from corn fields, relative to control sites for Rana clamitans in Quebec. This is believed to be a result of either acute or cumulative toxicity. None of the pesticides used in the investigation in Quebec were used in this investigation.

It is possible that application of pesticides directly to the frog may cause increased direct mortality or indirect mortality by slowing migration speed, thereby increasing the risk of predation, being runned down by machinery, or desiccation during the migration.

Indirect effects of pesticides

Indirect effects of pesticides on adult amphibian survival could be reduction of diversity and abundance of invertebrate food. Skylarks which depend upon foraging in open fields had a 38 % reduction in numbers of fledglings from sprayed fields, when compared to skylarks from unsprayed barley fields. The abundance of food was generally higher in the unsprayed fields and compared with sprayed fields immediately after spraying it was three times higher (Odderskær 1997). Dover et al (1990) found that the populations of some species of butterflies increased as a result of reduced pesticide input to headlands of cereal fields. Unsprayed headlands have a higher coverage, density, biomass and species diversity of weeds as well as a higher density of nontarget arthropods when compared to herbicide treated headlands (Chiverton & Sotherton 1991).

Serious threat to Bombina bombina

A decrease in survival due to cultivation is a serious threat to Bombina bombina, because it lays relatively few eggs compared to other frog species (Briggs 1993). Frog species of the genus Rana have normal yearly survivals from 30-60 % (Turner 1960, Briggs 1993), and mortality also occurs due to cultivation of fields for Rana sp. (Schneeweiss & Schneeweiss 1997). Due to lower reproductive potential, B. bombina needs higher adult survival than other frogs to ensure a stable development of a population.

 

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Version 1.0 October 2004, © Danish Environmental Protection Agency