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Temperature influences the toxicity of deltamethrin, chlorpyrifos and dimethoate to the predatory mite Hypoaspis aculeifer (Acari) and the springtail Folsomia candida (Collembola)
Ecotoxicology and Environmental Safety 140 (2017) 214–221
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Temperature influences the toxicity of deltamethrin, chlorpyrifos and dimethoate to the predatory mite Hypoaspis aculeifer (Acari) and the springtail Folsomia candida (Collembola)
MARK
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O.O. Jegedea,b, , O.J. Owojoria,c, J. Römbked,e a
Department of Zoology, Obafemi Awolowo University, Ile-Ife, Nigeria Toxicology Centre/Department of Soil Science, University of Saskatchewan, Saskatoon, SK, Canada c Department of Soil Science, University of Saskatchewan, Saskatoon, SK, Canada d ECT Oekotoxikologie GmbH, Floersheim, Germany e LOEWE Biodiversity and Climate Research Centre BiK-F, Frankfurt/Main, Germany b
A R T I C L E I N F O
A B S T R A C T
Keywords: Temperate Tropics Micro-arthropods Risk assessment Temperature
In order to assess the influence of temperature on pesticide toxicity to soil fauna, specimens of the predatory mite Hypoaspis aculeifer and the springtail Folsomia candida were exposed in artificial soil spiked with different concentrations of three pesticides (dimethoate, chlorpyrifos and deltamethrin) at 20 °C vs 28 °C for the mites and 20 °C vs 26 °C for the springtails. All tests were carried out according to OECD guidelines. In the mite tests, the toxic effects of dimethoate and chlorpyrifos on survival was about two orders of magnitude more at 28 °C than at 20 °C. Mite reproduction decreased in the tests with chlorpyrifos and deltamethrin by about four to five orders of magnitude at 28 °C than at 20 °C. (EC5028°C =1.42 and 2.52 mg/kg vs EC5020°C=6.18 and 10.09 mg/kg) In the collembolan tests, the toxicity of dimethoate on survival was higher at 26 °C than at 20 °C (LC5026°C =0.17 mg/ kg vs LC5020°C =0.36 mg/kg), while the opposite was detected for deltamethrin (LC5026°C =11.27 mg/kg vs LC5020°C =6.84 mg/kg). No difference was found in the test with chlorpyrifos. Effects of dimethoate and chlorpyrifos on reproduction were higher at 26 °C than at 20 °C (EC5026°C =0.11 and 0.018 mg/kg vs EC5020°C =0.29 and 0.031 mg/kg respectively), but in the case of deltamethrin the opposite was observed (EC5026°C =12.85 mg/kg vs EC5020°C =2.77 mg/kg). A preliminary risk assessment of the three pesticides at the two temperature regimes based on the Toxicity Exposure Ratio (TER) approach of the European Union, shows that in general there are few different outcomes when comparing data gained at different temperatures. However, in the light of the few comparisons made data gained in temperate regions should be used with caution in the tropics.
1. Introduction The use of pesticides in agriculture has increased substantially in the last fifty years (Popp et al., 2013). This upsurge in pesticide use is not limited to developed countries in the temperate regions, many developing countries in the tropical regions have embraced the practice (Hurtig et al., 2003; Afari-Sefa et al., 2015). Many pesticides are seldom strictly selective and they may also affect non target organisms (Martikainen, 1996). Therefore, the environmental risk of these pesticides has to be assessed, using ecotoxicological data gained in standardized tests. So far, most of these fate and effect tests are required and performed in countries located in temperate regions of the world. Consequently, the standard ecotoxicological laboratory tests of, for example, the European Union, are conducted at a temperature of 20 ± 2 °C (e.g.; ISO, 1999; OECD Organization for Economic Co-
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operation and Development, 2008 and OECD Organization for Economic Co-operation and Development, 2009). However, the data gained in these tests are often used also for the risk assessment of pesticides in tropical regions (Römbke et al., 2008), where testing requirements are less specified and average temperatures are often higher. At the same time it is well known that temperature can directly affect organisms exposed to these chemicals. Their metabolic rate, locomotion or feeding activity may be influenced by temperature thus affecting toxicant uptake and elimination as well as detoxification rates (e.g., Donker et al., 1998 and Garcia et al., 2011). In addition, temperature increase or decrease, also influences the fate of pesticides, mainly by altering their metabolization and degradation (e.g. Racke et al., 1997; Laabs et al., 2000 and Paraiba et al., 2003). Therefore, already about 20 years ago it has been proposed to adapt testing requirements to regional environmental conditions (Wiktelius et al.,
Corresponding author.
http://dx.doi.org/10.1016/j.ecoenv.2017.02.046 Received 5 September 2016; Received in revised form 25 February 2017; Accepted 27 February 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.
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Development, 2008, 2009). It contained 74.8% quartz, 20% kaolin clay, 5.2% sphagnum peat by dry weight and has a pH of 5.75 ± −0.5. The mixture was moistened to a maximum water holding capacity (WHC) of 60%.
1999). In the context of climate change, rising temperatures will even increase this problem (Noyes et al., 2009). The average global temperature is expected to increase by 1.4–5.8 °C in 2100 and this rise is expected to be more in the Temperate regions (FAO Food and Agriculture Organization, and IPCC, 2002, 2014). Few studies have investigated the effect of temperature on pesticide toxicity to soil organisms. For example, Garcia (2004) assessed the influence of temperature on toxicity of two fungicides (carbendazim and benomyl) and an insecticide (lambda-cyhalothrin) on the earthworm Eisenia fetida and the isopod Porcellionides pruinosus. He found lower toxicity for the fungicides and higher toxicity for the insecticide at tropical (28 °C) compared to temperate (20 °C) conditions. De Silva et al. (2009) studied the effects of temperature (20 vs 26 °C) on the toxicity of organophosphate pesticides to the earthworm E. andrei and found that toxicity was higher at higher temperatures. Recently, Bandow et al. (2014) observed increased susceptibility of the Collembolan Folsomia candida to a fungicide, pyrimethanil at a temperature of 26 °C compared to 20°C. Even interactions between effects of the insecticide lambda-cyhalothrin and soil moisture could be identified (Bandow et al., 2014). However, information on the toxicity of pesticides at different temperatures to soil organisms is still limited at best. For example, the influence of temperature on the pesticide toxicity to predatory mites has not been tested at all. Therefore, studies on the effect of pesticides on soil organisms at increased temperatures are necessary to correctly quantify their risk in the tropics. Insecticides are commonly used pesticides in many tropical regions and by design, are very potent in affecting different invertebrate species (Heong and Schoenly, 1998). Micro-arthropods (i.e. Acari and Collembola) are important inhabitants of agricultural soils, either as pests, as pest antagonists or as decomposers (i.e. influencing nutrient cycling via regulating microbial communities). These animals tend to be directly exposed to insecticide used in farmlands. The objectives of this study is to assess, the effects of temperature on the toxicity of three commonly and globally used insecticides,; a pyrethroid (deltamethrin) and two organophosphates (chlorpyrifos and dimethoate) on two standard micro-arthropods; Folsomia candida; a collembolan and Hypoaspis aculeifer, a gamasid mite.
2.3. Test substances Test chemicals used included three pesticides: dimethoate (an organophosphate, added as the formulation Bi 58, containing 400 g a.s./L, Compo GmbH, Germany) and chlorpyrifos (an organophosphate, added as the formulation Pestanal, purity: 99.9% CAS No: 2921-88-2, Sigma-Aldrich, Germany) and deltamethrin (a pyrethroid, added as the formulation Decisflussig, 2.8% active substance (a.s), Bayer Crop Science GmbH, Germany). The concentrations used in the individual tests were based on the results of range-finding-tests, covering a range between 0.1 and 1000 mg a.i./kg dw. 2.4. Test procedures Each treatment including the control had four replicates. Dimethoate and deltamethrin were added to the soil as aqueous solution in an amount of de-ionized water in order to reach 50% of the maximum WHC of the soil. For chlorpyrifos, the compounds were introduced to a portion (10%) of the soil, using acetone (1:2, v/w soil) as a carrier, and left in a fume hood for at least 24 h to allow the acetone solvent to evaporate after which it was mixed with remaining portion (90% of total soil). The soil was then subsequently moistened to the required moisture level of 50% of the maximum WHC. Solvent controls were also prepared for the test. Prepared soil treatments were introduced into the test vessels and the animals were introduced immediately afterwards. The mite test was performed as described in OECD Organization for Economic Co-operation and Development (2008). Ten adult female mites between ages 28–35 days were introduced in each test vessel containing 20 g dry weight soil. Each test vessel was covered with a paraffin film which was perforated to allow gaseous exchange. All test vessels were kept in climate chambers at 20 °C and 28 °C for 14 days and the test organisms were fed every 3 days with cheese mites. The temperature range was fixed based on information regarding the ecological preferences of this species (Jänsch et al., 2005). Moisture changes were assessed weekly by weighing the vessels and the corresponding water loss was replenished. After the 14th day of test, the test vessels were removed from the climate chamber and the mites were extracted using a modified Berlese-Tullgren apparatus. The extraction took about 2 days where the soils were exposed to light at temperatures of 25 °C for 12 h, 35 °C for 12 h and 45 °C for 24 h to ensure total recovery of the mites (OECD Organization for Economic Co-operation and Development, 2008). The Collembolan test was performed as described in OECD Organization for Economic Co-operation and Development (2009). Ten juvenile organisms (10–12 days) were introduced into glass vessels (50 ml), each containing 20 g of dry soil which had been mixed thoroughly with de-ionized water (controls) or the chemicals. The test vessels were kept in climate chambers at 20 °C and 26 °C for 28 days and food (5 mg of dry yeast) was added twice per week to each container. The temperature range was fixed based on information regarding the ecological preferences of this species (Jänsch et al., 2005). After 28 days, the test was ended. Each test vessel was emptied into a container filled with distilled water for extraction. The collembolans floated on the water surface. The adults and juveniles were counted separately in order to determine survival and reproduction. In order to facilitate the counting of the springtails, a black ink was added to the water to give a black background color against the white colors of the collembolans. The collembolans were counted under a light microscope.
2. Materials and methods 2.1. Test species The predatory mite Hypoaspis (Geolaelaps) aculeifer Canestrini (Acari: Laelapidae) was used in this study. The mites were taken from a culture bred in the laboratory of ECT Oekotoxikologie GmbH, Floersheim, Germany. They were kept at 20 °C with a photoperiod of 16:8 h, light: dark, in plastic containers lined with an 8:1 ratio of Plaster of Paris and charcoal. The substrate was moistened once a week with de-ionized water, and cheese mites (Tyrophagus putrescentiae) were added ad-libitum as a food source. Specimens used for the test were adult, 28–35 days old and were collected from a synchronised culture. The other test species was the springtail Folsomia candida (Collembola: Isotomidae). A culture of F. candida has been maintained for more than ten years at the laboratory of ECT Oekotoxikologie GmbH, Floersheim, Germany. They were reared in plastic containers lined with a medium of an 8:1 ratio of Plaster of Paris and charcoal maintained at 20 °C with a photoperiod of 16:8 h, light: dark, which was moistened weekly. A small amount of bread yeast was added ad libitum. Juvenile collembolans aged 10–12 days were used in the tests. 2.2. Test soil Artificial soil prepared according to the guidelines of the Organization for Economic Co-operation and Development was used in the experiments (OECD Organization for Economic Co-operation and Development, and OECD Organization for Economic Co-operation and 215
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3. Data assessment
In the tests with dimethoate (Fig. 1), at 28 °C percentage survival was higher than 100% in the control and also at 1.00 mg a.i./kg dw (not at 1.78 mg a.i./kg dw). The reason was that one could not differentiate the surviving adults from some rapidly maturing juveniles. Other juveniles in this case were differentiated by their lighter brown color but no juvenile was white as it is supposed to be. At 28 °C, no mites survived at the two higher concentrations, which happened at 20 °C only at the highest concentration. In general, survival was clearly lower at 28 °C than at 20 °C, as indicated by the LC50 values which do not overlap (Table 2). Effects of dimethoate on mite reproduction were similar at both temperatures (see EC50/EC 10 values; Table 2), but the number of juveniles was much lower at all concentrations at 28 °C compared to 20 °C. At 28 °C no juveniles were found starting from 5.62 mg/kg concentration and a strong decline in reproduction was found for the same concentration at 20 °C. For chlorpyrifos, at both temperatures very few mites survived at the highest concentration of 10 mg a.i./kg dw (Fig. 1). The higher toxicity of chlorpyrifos on survival at 28 °C than 20 °C is indicated by the LC50 values which differ by a factor of about two (Table 2). At 28 °C, significant effects on reproduction started at 0.32 mg a.i./kg dw, whereas at 20 °C significant effects (ANOVA, p < 0.05) on reproduction were found quite abruptly only at 10 mg a.i./kg dw(Fig. 1). Again, absolute juvenile numbers at all concentrations were much lower at the higher temperature. The EC50 values did not differ much from the LC50 values at the respective temperature, but they were by a factor of about four lower at 28 °C than at 20 °C (Table 2). For deltamethrin, there was no significant effect (p > 0.05) on survival up till 10 mg a.i./kg dwat both 28 °C and 20 °C (Fig. 1). At both temperatures, only very few individuals survived at 32 mg a.i./kg dw; i.e. there was a sharp increase in toxicity. At 28 °C, survival was higher than 100% in the control and two concentrations (1 and 3.2 mg a.i./kg dw, but at 0.32 mg a.i./kg dw the opposite was found), again indicating quicker development at the higher temperature. The LC50 values did not differ at the two temperatures (Table 2). At 20 °C, juvenile numbers did not differ between the control and 3.2 mg a.i./kg dw, but decreased by ca. 50% at 10 mg a.i./kg dw and were zero at the highest concentration of 32 mg a.i./kg dw. At 28 °C° toxic effects on mite reproduction decreased at an (almost) constant rate over the whole concentration range. Consequently, the EC50 values were by a factor of four higher at 20 °C than at 28 °C – and the EC10 values differed even by a factor of 30 (Table 2). In summary, out of the nine comparisons (three endpoints and three pesticides) which are shown here, toxicity increased with increasing temperature in six cases (Higher sensitivity at 28 °C. LC50 of dimethoate, chlorpyrifos, EC50 of chlorpyrifos and deltamethrin, EC10 of chlorpyrifos and deltamethrin.). In the other three cases (Lower sensitivity at 28 °C LC50 of deltamethrin, EC50 of dimethoate and EC10 of dimethoate.), there was no difference in the LC/EC values based on the observation whether there was an overlap of the confidence intervals or not. Not surprisingly, this overlap was most pronounced for the EC10 values, i.e. in all three cases. There is no clear correlation between the test chemical and the toxicity pattern in relation to the change in temperature.
3.1. Statistics Nominal concentrations of pesticides were used for calculations. The LC50 values were calculated using mortality data and the Trimmed Spearman-Kärber Program version 1.5, because a good fit was not obtained with probit analysis. The EC50and EC10 values of the pesticides for reproduction were calculated using the Linear Interpolation Method (USEPA, 1993) or non-linear regression methods (logistic 3-parameter) methods respectively, depending on which model gave a higher adjusted r2 value. In cases where the Linear Interpolation method was adopted for analysis, the expanded confidence limits were used because the number of replicates per treatment was less than seven (Environment Canada, 2005). Differences between the reproduction EC50 values on one hand and the LC50 values on the other hand for the two temperature regimes were ascertained when confidence limits did not overlap. The effect of increasing pesticide concentration on survival and reproduction of the collembolans and mites was assessed with a one way analysis of variance (ANOVA). The tukey post-hoc comparison was used to check where the differences lied. To test the direct effect of temperature on survival and reproduction of the mites and collembolans in control treatments, student's t-test was used. 3.2. Preliminary risk assessment of pesticides at tropical temperatures A preliminary risk assessment of the three pesticides was carried out by estimating the risk using the Toxicity Exposure Ratio (TER) as described by Römbke et al. (2008), i.e. following the rules used for the registration of pesticides in the European Union (EC (European Commission) and EC (European Commission), 2002, 2009). For the acute risk, the TERacute is the ratio of the LC50 to the predicted environmental concentration (PEC). For all pesticides and soils a single application and the highest normal application rate (deltamethrin: 224 g/ha (EFSA (European Food Safety Authority), 2010); dimethoate: 748 g/ha (EFSA (European Food Safety Authority), 2013); chlorpyrifos: 648 g/ha (EFSA (European Food Safety Authority), 2014) was used when calculating the PEC (based on a soil depth of 5 cm and a soil bulk density of 1.5) (Table 1). If the TERacute ≥10, a low acute risk is indicated. For the chronic risk, the TERchronic is the ratio of the NOEC or EC10 to the PEC. The EC10 was used instead of the NOEC because it is relatively more precise (Warne and van Dam, 2008). If the TERchronic > 5, a low chronic risk is indicated. 4. Results 4.1. Survival and reproduction of Hypoaspis aculeifer In the control vessels, validity criteria (≥80% survival) and (≤30% coefficient of variation) were met in all survival tests (OECD Organization for Economic Co-operation and Development, 2008). In general, the number of juveniles produced in the controls was significantly lower (t-test, p < 0.05) at 28 °C than at 20 °C.
Table 1 Calculation of the predicted environmental concentration (PEC) for the three pesticides used (all concentrations refer to dry weight).
Soil depth (d) Bulk density (D) Vol (V)=d x length (l) x breadth (b) Mass (M)=D X V Application rate (a.r) a.r converted to g/m2(1 ha = 10,000 m2) PEC in mg/kg (Given 1 m2=75 kg of soil) PEC
Dimethoate
Chlorpyrifos
Deltamethrin
0.05 m 1500 kg/m3 0.05×1×1=0.05 m3 0.05×1500=75 kg 748 g/ha 748/10,000=0.0748 g/m2 0.0748/75=1.00 mg/kg 1.00 mg/kg
0.05 m 1500 kg/m3 0.05 × 1×1 = 0.05 m3 0.05×1500=75 kg 648 g/ha 648/10,000=0.0648 g/m2 0.0648/75=0.86 mg/kg 0.86 mg/kg
0.05 m 1500 kg/m3 0.05×1×1=0.05 m3 0.05×1500=75 kg 224 g/ha 224/10,000=0.0224 g/m2 0.0224/75=0.30 mg/kg 0.30 mg/kg
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Fig. 1. Mean ( ± SE, n=4) percentage survival (left) and number of juveniles (right) produced by the predatory mite Hypoaspis aculeifer exposed to dimethoate, chlorpyrifos and deltamethrin in OECD soil under stable laboratory conditions at 20 °C and 28 °C. Surv 20= Survival at 20 °C, Surv 28= Survival at 28 °C, Repro 20= Reproduction at 20 °C, Repro 28= Reproduction at 28 °C.
nificantly between the two temperatures, but were less than a factor of two lower than the EC50 values. For chlorpyrifos, comparable effect patterns on reproduction as for dimethoate were observed at both temperatures (Fig. 2). The LC50 and EC50 were similar at both temperatures, but in absolute terms at very low concentrations (Table 3). Significant effects (ANOVA, p < 0.05) on reproduction were found at even the lowest concentrations tested, with very few or no springtails observed at the two highest concentrations (0.05 and 0.1 mg a.i/kg dw). This reflected in the EC50 and EC10 values because they were all very low (Table 3). For deltamethrin, toxic effects were lower at 26 °C than at 20 °C which was evident in the percentage survival recorded at both temperatures. This fact is also visible in the LC50 values which were by a factor of two higher at 26 °C (Table 3). This pattern of toxicity was also observed for reproduction, i.e. toxic effects of deltamethrin were lower at 26 °C than at 20 °C (Fig. 2). Consequently, this fact is also reflected by the EC50 at 26 °C (higher by a factor of four) and EC10 at 26 °C (higher even by a factor ten) values. In the tests with dimethoate toxicity always increased. In the tests with chlorpyrifos, toxicity based on survival was similar at both
4.2. Survival and reproduction of Folsomia candida The validity criteria (> 80% survival, > 100 juveniles, < 30% coefficient of variation) for Collembola were met for all tests (OECD Organization for Economic Co-operation and Development, 2008). Generally, there was no significant difference (p < 0.05) in the % survival at 26 and 20 °C in the controls but the number of juveniles produced in the controls was significantly lower (p < 0.05) at 26 °C than at 20 °C, with one exception: in the tests with chlorpyrifos the number of juveniles produced at 26 °C was generally higher than the ones produced at 20 °C but the difference was not statistically significant (p > 0.05). At both temperatures, effects of dimethoate on the survival of F. candida became significant from 0.4 mg a.i./kg dw (Fig. 2). With the exception of few individuals at 0.4 mg ai.i/kg dw and 20 °C no springtails survived at higher concentrations from 0.4 mg a.i/kg dw. Toxic effects on the survival of the collembolans were higher at 26 °C than at 20 °C as confirmed by the LC50 values, which differed by a factor of two (Table 3). At both temperatures the EC50 values were only slightly lower than the LC50 values. The EC10 values differed sig-
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Table 2 Comparison of the median lethal concentration (LC50) values (a), the median effective concentration (EC50) values (b), and the 10% effective concentration (EC10) values (c) with confidence intervals for the effects on survival and reproduction of the Predatory mite H. aculeifer exposed to dimethoate, chlorpyrifos and deltamethrin in OECD artificial soil at 20 °C and 28 °C.
Table 3 A comparison of the median lethal concentration (LC50) values (a) median effective concentration (EC50) values (b) and 10% effective concentration (EC10) values with confidence interval for the effects on survival and reproduction of F. candida exposed to dimethoate, chlorpyrifos and deltamethrin in OECD artificial soil at 20 °C and 26 °C. Pesticides
Pesticides
20 oC a) LC50 Dimethoate(mg/kgdw) Chlorpyrifos(mg/kgdw) Deltamethrin(mg/kgdw) b) EC50 Dimethoate(mg/kgdw) Chlorpyrifos(mg/kgdw) Deltamethrin(mg/kgdw) c) EC10 Dimethoate(mg/kgdw) Chlorpyrifos(mg/kgdw) Deltamethrin(mg/kgdw)
Temperature
Temperature
4.60 (3.90 − 5.00) 5.50 (5.10 − 5.80) 16.30 (13.60 − 19.40)
4.00 (3.55 − 4.45) 6.18 (−9.94 − 22.30) 10.09 (9.11 − 11.07)
2.69 (2.17 − 3.20) 5.09 (−11.05 − 21.24) 7.51 (−8.08 − 23.11)
20 °C
26 °C
a) LC50 Dimethoate(mg/kgdw) Chlorpyrifos(mg/kgdw) Deltamethrin(mg/kgdw)
0.36 (0.32 −0.40) 0.04 (0.03–0.05) 6.84 (5.93–7.89)
0.17 (0.15 − 0.19) 0.04 (0.03–0.04) 11.27 (8.52–14.90)
b) EC50 Dimethoate(mg/kgdw) Chlorpyrifos(mg/kgdw) Deltamethrin(mg/kgdw)
0.29 (0.26 − 0.33) 0.031 (0.023 − 0.038) 2.77 (1.04 − 4.49)
0.11 (0.09 − 0.13) 0.018 (0.01 − 0.024) 12.85 (7.72 − 17.98)
c) EC10 Dimethoate(mg/kgdw) Chlorpyrifos(mg/kgdw) Deltamethrin(mg/kgdw)
0.21 (0.17 − 0.25) 0.016 (0.005 − 0.26) 0.57 (−0.29 − 1.43)
0.055 (0.035 − 0.075) 0.005 (0.001 − 0.009) 5.40 (−2.26 − 13.07)
28 oC
2.82 (2.50 – 3.10) 2.41 (1.7 – 3.31) 17.50 (16.40 – 18.70)
3.39 (0.86 − 5.92) 1.42 (0.94 − 1.91) 2.52 (0.89 − 4.17)
2.90 (0.34 − 5.46) 0.71 (0.23 − 1.20) 0.24 (0.1 − 0.59)
Fig. 2. Mean ( ± SE, n=4) percentage survival (Left) and number of juveniles (right) produced by the collembolan Folsomia candida exposed to dimethoate, chlorpyrifos and deltamethrin in OECD soil under stable laboratory conditions at 20 °C and 26 °C. Surv 20= Survival at 20 °C, Surv 26= Survival at 26 °C, Repro 20= Reproduction at 20 °C, Repro 26= Reproduction at 26 °C.
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Table 4 Risk assessment of dimethoate, chlorpyrifos and deltamethrin at tropical temperatures(28 °C/26 °C), based on the comparison of acute (LC50) and chronic (EC10) effect values determined in laboratory tests with two micro-arthropods with predicted environmental concentrations (PECs) based on recommended application rates in agriculture (always one application of the respective pesticide). A risk is indicated (* and bold) when the Toxicity Exposure Ratio (TER) values are below ≤10 (acute tests) or ≤5 (chronic tests) (EC (European Commission), 2002). All concentrations are given as mg ai/kg dw. a. Hypoaspis aculeifer Pesticide
Rate a.i. (g/ha)
PEC (mg/kg)
20 °C LC50
20 °C Acute TER
28 °C LC50
28 °C Acute TER
20 °C EC10
20 °C Chronic TER
28 °C EC10
28 °C Chronic TER
Dimethoate Chlorpyrifos Deltamethrin
748 648 224
1.00 0.86 0.30
4.60 5.50 16.30
4.60* 6.40* 54.33
2.80 2.40 17.50
2.80* 2.79* 58.33
2.69 5.09 7.51
2.69* 5.92 25.23
2.90 0.71 0.24
2.90* 0.83* 0.80*
b. Folsomia candida Pesticide
Rate a.i. (g/ha)
PEC (mg/kg)
20oCLC50
20oC Acute TER
26oC LC50
26oC Acute TER
20oC EC10
20oC Chronic TER
26oC EC10
26oC Chronic TER
Dimethoate Chlorpyrifos Deltamethrin
748 648 224
1.00 0.86 0.30
0.36 0.04 6.84
0.36* 0.05* 22.80
0.17 0.04 11.27
0.17* 0.05* 37.57
0.21 0.016 0.57
0.21* 0.019* 1.90*
0.055 0.005 5.40
0.055* 0.006* 18.00
Acute TER = LC50/PEC; Chronic TER = EC10/PEC.a.i (g/ha) = active ingredient in gram/hectare
that these tests were performed slightly out of the range of preference of H. aculeifer while still be in the range of tolerance (Jänsch et al., 2005). However, since the results even in these two tests with relatively low number of juveniles did follow a dose-response-curve and other validity criteria such as the required range of variation were fulfilled we consider the data as relevant for our study. In future tests and referring to the information gained in our tests, it might be better to perform mite tests mimicking tropical conditions also at 26 °C (see also next paragraph). A temperature of 28 °C representative of tropical temperatures against a temperate temperature of 20 °C was initially chosen for the tests with springtails (F. candida) too. However, according to information collected by Jänsch et al. (2005) a temperature of 26 °C is likely the upper limit of the range of tolerance for F. candida. Therefore, the temperature for the collembolan tests was lowered down to 26 °C. Since 26 °C is still within the range of tropical temperatures generally known to be within 25–35 °C, the results of this study could still be considered to be relevant for tropical environment.
temperatures but toxicity based on reproduction increased at 28 °C than at 20 °C. Deltamethrin caused either no difference in toxicity (EC50) or toxicity even decreased with increasing temperature. In summary, out of the nine comparisons (three endpoints and three pesticides) which are shown here, toxicity increased with increasing temperature in five cases, in three cases toxicity decreased and in one case there was no difference in toxicity. 4.3. Preliminary risk assessment of pesticides at tropical temperatures Referring to the rules of the European Union for the risk assessment of pesticides (EC (European Commission) (2002)), a risk for soil microarthropods cannot be excluded when comparing recommended application rates with effects determined in laboratory tests with the species H. aculeifer and F. candida (Tables 4a & 4b). In the tests with the mite H. aculeifer and dimethoate an acute and chronic risk is indicated at both temperatures. Almost the same result was found in the tests with chlorpyrifos, with one exception: no risk was found when comparing the chronic test results gained at 20 °C with the PEC. In the tests with deltamethrin a risk was found only in the chronic test at 28 °C. In summary, for the three insecticides clearly different outcomes in the risk assessment procedure were determined and a risk was found more often at 28 °C (five times) than at 20 °C (three times). In the tests with the springtail F. candida and dimethoate acute and chronic risks are also indicated at both temperatures. The same outcome was found in the tests with chlorpyrifos. In contrast to the other species, however, in the tests with deltamethrin a risk was only indicated at the lower temperature (20 °C). Thus, this species reacts slightly more sensitive than the mites – and also slightly more often at the lower (five times) than the higher (four times) temperature.
5.2. Interaction of temperature and pesticides on toxicity to microarthropods This study shows that temperature influenced the toxicity of the three tested pesticides to the two micro-arthropod species (H. aculeifer and F. candida). The organophosphates dimethoate and chlorpyrifos were highly toxic to both species at both temperatures. In fact, F. candida was clearly more sensitive than H. aculeifer (with one exception: the test with deltamethrin at 28 °C) – a relationship which has been found already for many other chemicals (Huguier et al., 2015). In contrast, the third insecticide, the pyrethroid deltamethrin, was less toxic (especially for F. candida). Interestingly, this difference is not very pronounced at 20 °C, but clearly at 26 °C. It might be that the concentration of deltamethrin decreased more quickly at the higher temperature, since in the literature the mean degradation time (DT50) under temperate conditions is given as 35 days, while in Brazilian field sites a much lower DT50 value of about 12.4 days has been reported at higher, but not specified temperatures (Laabs, and Römbke et al., 2002, 2008). However, physiological reasons may also play a role whereby collembolans possibly were able to metabolize and eliminate deltamethrin faster at high temperatures. Looking at the absolute toxicity values, it seems that, independent from the endpoint (mortality, reproduction), toxicity increased by a factor of two to four with increasing temperature. However, these differences were not always significant because of the variability of the
5. Discussion 5.1. Temperature effect on microarthropods Usually, tests with mites and springtails are performed according to standard methods (OECD Organization for Economic Co-operation and Development, 2008, 2009), i.e. at a temperature of 20 ± 2 °C. We could show that the same tests with the same species can also be performed at 28 °C (mites) and 26 °C (springtails) – and with the exception of two mite reproduction tests in which one validity criterion was not met (i.e. less than 100 juveniles were found in the controls) all of them were valid. In the light of the fact that juvenile numbers were always lower in all tests running at 28 °C compared to those performed at 20 °C it seems 219
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more frequently (Joose and Buker, 1979). The difference in response of F. candida and H. aculeifer to deltamethrin at high and low temperatures could only be attributed to species differences. The preliminary risk assessment showed that elevated temperature, typical of tropical areas, could enhance the risk of these pesticides to mites and collembolans in the soil. However, the differences in toxicity were often not high enough in order to increase the risk considerably. In addition, these insecticides, might not be the best substances for studying this effect, because they are already very toxic at lower temperatures such as 20 °C. In addition, one may ask whether the risk classes (i.e. the trigger values of 10 and 5) are appropriate. However, in any case it is unlikely that higher temperatures cause a decrease in toxicity and, thus, risk – this happened only twice in the tests with F. candida – in all other cases toxicity and risk either stayed similar or increased. There is another factor to be considered in this discussion: In the present study, just one application was used to calculate the PEC. However, most of the time, farmers in the developing tropical countries spray their farmlands more than once per season, independently from respective application rules. In such cases, the PEC will increase, meaning that the risks described here will increase. Already now, a high risk may occur when using the two organophosphates – and also deltamethrin cause a risk at higher temperatures (at least for mites).
individual endpoints. This pattern was observed for the OP pesticides (dimethoate and chlorpyrifos) for both species and for the pyrethroid (deltamethrin) in tests with H.aculeifer. The toxicity of deltamethrin at high temperature did not follow a specific pattern; both increases and decreases could be observed depending on the species. Some studies (Weston, and Harwood et al., 2009, 2009) have shown that pyrethroids tend to be more toxic to organisms at lower temperatures. For example, there was a 2–13 fold increase in toxicity to cockroaches, tobacco budworms, cabbage looper, potato beetles at about 10 °C decrease in temperature (Harwood et al., 2009). Pyrethroids are sodium channel modulators and as such, at reduced temperatures, there is an increased stability of open-modified sodium channels which further prolongs the duration of sodium influx, thereby increasing the vulnerability of the nervous system to toxic effect. This trend was demonstrated in our study with F. candida but it is not exactly clear why this trend was not found for the mite H. aculeifer in this study. More generally speaking, at higher temperatures more or other metabolites could occur, meaning uptake, distribution, transport and metabolization of chemicals within the organisms might be changed (Howe et al., 1994), processes which are also discussed in the context of climate change (Noyes et al., 2009). However, for the scope of this study, we wanted to investigate toxicity to mites and collembolans at high and low temperatures and not specifically on the mechanism of toxicity. In summary the outcome of some of these tests was not really expected, since referring to the literature, effects should increase at higher temperatures. For example, in a recent study (Bandow et al., 2014), increasing toxicity of the fungicide pyrimethanil to the collembolan F. candida was observed at a temperature of 26 °C when compared to the results gained at 20 °C. However, this study shows that toxicity may be species or chemical specific. A study such as this with tropical species could have given more information. But according to the review of Huguier et al. (2015), no such studies were performed with tropical mites so far. The only exception refers to a moss mite, Archegozetes longisetosus (Oribatida; Trhypochthoniidae), which was tested with cadmium at 30 °C (Seniczak et al., 2009). So, studies on the effects of pesticides to other arthropods were also reviewed as well as experiences with other invertebrates exposed to our test chemicals. Garcia (2004) observed increased toxicity of the pyrethroid insecticide lambda-cyhalothrin to the isopod Porcellionides pruinosus (and the earthworm Eisenia fetida) at 28 °C when compared to 20 °C. This finding was supported by De Silva et al. (2009) who reported increased toxicity of the organophosphate pesticide chlorpyrifos to the earthworm Eisenia andrei at 26 °C than at 20 °C. Römbke et al. (2008) observed that the pyrethroids deltamethrin and lambda-cyhalothrin did not pose risk to earthworms under tropical conditions in Central Amazonian soils, in contrast to the organophosphate parathion methyl. In that study, the same EU approach was used to assess the risk of pesticides, but these authors incorporated the median disappearance time (DT50) as well as the number of pesticide applications within one season. However, this kind of data can only be gathered site-specifically, i.e. we could not include these refinements in our study. Our results here are supported by literature data, indicating the same trends of increasing toxicity at increasing temperatures. Because only few studies of this kind had been performed with H. aculeifer and F. candida so far, we also compared the results of our studies with those performed with other soil invertebrates which also could be exposed to pesticides. The other pattern of toxicity was reduced toxicity of pesticides at increased temperature, as found for deltamethrin on F. candida in which toxic effect on survival and reproduction of F. candida was higher at 20 °C than at 26 °C by almost by a factor of 2. This observation could be due to degradation of deltamethrin which is faster at higher temperatures of 25 °C than at 20 °C in the laboratory under standardized conditions (Bayer Crop Science, 2009). Also, at high temperatures, excretion of toxic substances by molting and intestine renewal occur
6. Conclusions The study has shown that changes in temperature can influence the toxicity of chlorpyrifos, dimethoate and deltamethrin to soil microarthropods and re-emphasize that temperature is an important abiotic factor which is necessary for proper soil functioning. The study also showed that species and pesticide differences could influence the toxic response when temperature changes. As in the case of Folsomia candida, it was less sensitive to deltamethrin at high temperatures compared to Hypoaspis aculeifer which was sensitive to deltamethrin at higher temperature. Based on the data from this study where tests were carried out at temperate (20 °C) and tropical temperatures (26 and 28 °C), it could be deduced that toxicity of pesticides are generally higher under tropical conditions. For the purpose of risk assessment, the toxicity were not high enough to increase risk remarkably. However, due to temperature differences asides other climatic conditions extrapolation of data from temperate climate to tropical climate should still be used with caution. It is also noteworthy to incorporate the disappearance time for the pesticides for future work and use a tropical soil for this kind of study as in Römbke et al. (2008). This study also provides some preliminary data that could show how previous risk assessment evaluations might be inaccurate in the context of global warming. Acknowledgments We would like to thank Adam Scheffczyk (ECT Oekotoxikologie GmbH) who was involved with helping to perform these tests. References Afari-Sefa, V., Asare-Bediako, E., Kenyon, L., Micah, J.A., 2015. Pesticide use practices and perceptions of vegetable farmers in the cocoa belts of the Ashanti and Western regions of Ghana. Adv. Crop Sci. Technol. 3, 174. http://dx.doi.org/10.4172/23298863.1000174. Bandow, C., Karau, N., Roembke, J., 2014. Interactive effects of pyrimethanil, soil moisture and temperature on Folsomia candida and Sinella curviseta (Collembola). Appl. Soil Ecol. 81, 22–29. Bayer Crop Science, 2009. In: Proceedings of the Pyrethroid Scientific Forum 2009 (2) Vol 62. De Silva, C.S.M., C.S., Van Gestel, C.A.M., 2009. Development of an alternative artificial soil for earthworm toxicity testing in tropical countries. Appl. Soil Ecol. 43, 170–174 (2009). Donker, M.H., Abdel-Lateif, H.M., Khalil, M.A., Bayoumi, B.M., Van Straalen, N.M., 1998. Temperature, physiological time, and zinc toxicity in the isopod Porcellio scaber. Environ. Toxicol. Chem. 17, 1558–1563.
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Joose, E.N.G., Buker, J.B., 1979. Uptake excretion Lead. litter Dwell. Collembola Environ. Pollut. 18, 235–240. Laabs, V., Amelung, W., Pinto, A., Zech, W., 2002. Fate of pesticides in tropical soils of Brazil under field conditions. J. Environ. Qual. 31, 256–268. Laabs, V., Amelung, W., Pinto, A., Altstaedt, A., Zech, W., 2000. Leaching and degradation of corn and soybean pesticides in an Oxisol of the Brazilian Cerrados. Chemosphere 41, 1441–1449. Martikainen, E., 1996. Toxicity of dimethoate to some soil animal species in different soil types. Ecol. Environ. Saf. 33, 128–136. Noyes, P.D., McElwee, M.K., Miller, H.D., Clark, B.W., Van Tiem, L.A., Walcott, K.C., Erwin, K.N., Levin, E.D., 2009. The toxicology of climate change: environmental contaminants in a warming world. Environ. Int. 35, 971–986. OECD (Organization for Economic Co-operation and Development), 2008. OECD Guidelines for the testing of chemicals 226. Predatory mite Hypoaspis aculeifer reproduction test in soil. OECD/OCDE Code 226. Adopted in 2008. OECD(Organization for Economic Co-operation and Development), 2009. OECD Guidelines for the testing of Chemicals 232. Collembolan reproduction test in soil. Organization for Economic Co-operation and Development, Paris. Paraiba, L.C., Cerdeira, A.L., Silva, E.F., Martins, J.S., Coutinho, H.L.C., 2003. Evaluation of soil temperature effect on herbicide leaching potential into groundwater in the Brazilian Cerrado. Chemosphere 53, 1087–1095. Popp, J., Pető, K., Nagy, J., 2013. Pesticide productivity and food security: a review. Agron. Sustain. Dev. 33, 243–255. Racke, K.D., Skidmore, M.W., Hamilton, D.J., Unsworth, J.B., Miyamoto, J., Cohen, S.Z., 1997. Pesticide fate in tropical soils. Pure Appl. Chem. 69, 1349–1371. Römbke, J., Waichman, A.V., Garcia, M.V.B., 2008. Risk assessment of pesticides for soils of the central Amazon, Brazil: Comparing outcomes with temperate and tropical data. Int. Environ. Assess. Manag. 4, 94–104. Seniczak, A., Ligocka, A., Seniczak, S., Paluszak, Z., 2009. The influence of cadmium on life-history parameters and gut microflora of Archegozetes longisetosus (Acari: Oribatida) under laboratory conditions. Exp. Appl. Acarol. 47, 191–200. USEPA, 1993. A Linear Interpolation Method for sub-lethal toxicity: the inhibition concentration (ICp) Approach (Version 2.0) National Effluent Toxicity Assessment Center Technical Report 03-93. Warne, M.J., van Dam, R., 2008. NOEC and LOEC data should no longer be generated or used. Australas. J. Ecotoxicol. 14 (2008), 1–5. Weston, D.P., You, J., Harwood, A.D., Lydy, M.J., 2009. Whole sediment toxicity identification evaluation tools for pyrethroid insecticides: III. Temperature manipulation. Environ. Toxicol. Chem. 28, 173–180. http://dx.doi.org/10.1897/08143.1. Wiktelius, S., Chiverton, P.A., Meguenni, H., Bennaceur, M., Ghezal, F., Umeh, E.D.N., Egwuatu, R.I., Minja, E., Makusi, R., Tukahirwa, E., Tinzara, W., Deedat, Y., 1999. Effects of insecticides on non-target organisms in African agroecosystems: a case for establishing regional testing programmes. Agric. Ecosyst. Environ. 75 (1999), 121–131.
EC (European Commission), 2002. Draft Working Document. Guidance Document on Terrestrial Ecotoxicology Under Council Directive 91/414/EEC. SANCO/10329/2002 rev 2 final. EC (European Commission), 2009. Regulation (EC) No 1107/2009 of the European parliament and the council of 21 October 2009 concerning the placing of plant protection products on the market and repealing Council directives 79/117/EEC and 91/414/EEC. Off. J. Eur. Union L 309, 1–50. EFSA (European Food Safety Authority), 2010. Modification of the existing MRL for deltmethrin in potatoes. Eur. Food Saf. Auth., Parma, Italy EFSA J. 8 (11), 1900. EFSA (European Food Safety Authority), 2013. Conclusion on the peer review of the pesticide risk assessment of confirmatory data submitted for the active substance dimethoate. EFSA J. 11 (7) (3233, 36 pp). EFSA (European Food Safety Authority), 2014. Conclusion on the peer review of the pesticide human health risk assessment of the active substance chlorpyrifos. EFSA J. 12 (4) (3640, 34 pp). Environment Canada, 2005. Guidance document: Statistical methods for environmental toxicity tests. Environmental Protection Series, EPS1/RM/46. Ottawa, ON. FAO (Food and Agriculture Organization), 2002. Towards 2015/2030. World Agriculture: Summary Report. Rome 2002. Garcia, M., Scheffczyk, A., Garcia, T., Römbke, J., 2011. The effects of the insecticide lambda-Cyhalothrin on the earthworm Eisenia fetida under experimental conditions of tropical and temperate regions. Environ. Pollut. 159, 398–400. Garcia, M.V., 2004. Effects of pesticides on soil fauna: development of ecotoxicological test methods for tropical regions. Ecol. Dev. (Series No 19. ZEF Bonn). Harwood, A.D., You, J., Lydy, M.J., 2009. Temperature as a toxicity identification evaluation tool for pyrethroid insecticides: Toxicokinetic confirmation. Environ. Toxicol. Chem. 28, 1051–1058. Heong, K.L., Schoenly, K.G., 1998. Impact of insecticides on herbivore-natural enemy communities in tropical rice ecosystems. In: Haskell, P.T., McEwen, P. (Eds.), Ecotoxicology: Pesticides and Beneficial Organisms. Chapman & Hall, London, pp. 381–403. Howe, G.E., Marking, L.L., Bills, T.D., Rach, J.J., Mayer, F.L., 1994. Effects of water temperature and pH on toxicity of terbufos, trichlorfon, 4-nitrophenol and 2,4dinitrophenol to the amphipod Gammarus pseudolimnaeus and rainbow trout (Oncorhynchus mykiss). Environ. Toxicol. Chem. 13, 51–66. Huguier, P., Manier, N., Owojori, O.J., Bauda, P., Pandard, P., Römbke, J., 2015. The use of soil mites in ecotoxicology: a review. Ecotoxicology 24, 1–18. Hurtig, A.K., San Sebastián, M., Soto, A., Shingre, A., Zambrano, D., 2003. Pesticide use among farmers in the Amazon basin of Ecuador. Arch. Environ. Health 58, 223–228. IPCC, 2014. Climate change 2014. Synthesis Report. Edited by C.W Team, R.K Pachauri and L.A Meyer. Geneva, Switerland. Jänsch, S., Amorim, M.J.B., Römbke, J., 2005. Identification of the ecological requirements of important terrestrial ecotoxicological test species. Environ. Rev. 13, 51–83.
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Temperature influences the toxicity of deltamethrin, chlorpyrifos and dimethoate to the predatory mite Hypoaspis aculeifer (Acari) and the springtail Folsomia candida (Collembola)
MARK
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O.O. Jegedea,b, , O.J. Owojoria,c, J. Römbked,e a
Department of Zoology, Obafemi Awolowo University, Ile-Ife, Nigeria Toxicology Centre/Department of Soil Science, University of Saskatchewan, Saskatoon, SK, Canada c Department of Soil Science, University of Saskatchewan, Saskatoon, SK, Canada d ECT Oekotoxikologie GmbH, Floersheim, Germany e LOEWE Biodiversity and Climate Research Centre BiK-F, Frankfurt/Main, Germany b
A R T I C L E I N F O
A B S T R A C T
Keywords: Temperate Tropics Micro-arthropods Risk assessment Temperature
In order to assess the influence of temperature on pesticide toxicity to soil fauna, specimens of the predatory mite Hypoaspis aculeifer and the springtail Folsomia candida were exposed in artificial soil spiked with different concentrations of three pesticides (dimethoate, chlorpyrifos and deltamethrin) at 20 °C vs 28 °C for the mites and 20 °C vs 26 °C for the springtails. All tests were carried out according to OECD guidelines. In the mite tests, the toxic effects of dimethoate and chlorpyrifos on survival was about two orders of magnitude more at 28 °C than at 20 °C. Mite reproduction decreased in the tests with chlorpyrifos and deltamethrin by about four to five orders of magnitude at 28 °C than at 20 °C. (EC5028°C =1.42 and 2.52 mg/kg vs EC5020°C=6.18 and 10.09 mg/kg) In the collembolan tests, the toxicity of dimethoate on survival was higher at 26 °C than at 20 °C (LC5026°C =0.17 mg/ kg vs LC5020°C =0.36 mg/kg), while the opposite was detected for deltamethrin (LC5026°C =11.27 mg/kg vs LC5020°C =6.84 mg/kg). No difference was found in the test with chlorpyrifos. Effects of dimethoate and chlorpyrifos on reproduction were higher at 26 °C than at 20 °C (EC5026°C =0.11 and 0.018 mg/kg vs EC5020°C =0.29 and 0.031 mg/kg respectively), but in the case of deltamethrin the opposite was observed (EC5026°C =12.85 mg/kg vs EC5020°C =2.77 mg/kg). A preliminary risk assessment of the three pesticides at the two temperature regimes based on the Toxicity Exposure Ratio (TER) approach of the European Union, shows that in general there are few different outcomes when comparing data gained at different temperatures. However, in the light of the few comparisons made data gained in temperate regions should be used with caution in the tropics.
1. Introduction The use of pesticides in agriculture has increased substantially in the last fifty years (Popp et al., 2013). This upsurge in pesticide use is not limited to developed countries in the temperate regions, many developing countries in the tropical regions have embraced the practice (Hurtig et al., 2003; Afari-Sefa et al., 2015). Many pesticides are seldom strictly selective and they may also affect non target organisms (Martikainen, 1996). Therefore, the environmental risk of these pesticides has to be assessed, using ecotoxicological data gained in standardized tests. So far, most of these fate and effect tests are required and performed in countries located in temperate regions of the world. Consequently, the standard ecotoxicological laboratory tests of, for example, the European Union, are conducted at a temperature of 20 ± 2 °C (e.g.; ISO, 1999; OECD Organization for Economic Co-
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operation and Development, 2008 and OECD Organization for Economic Co-operation and Development, 2009). However, the data gained in these tests are often used also for the risk assessment of pesticides in tropical regions (Römbke et al., 2008), where testing requirements are less specified and average temperatures are often higher. At the same time it is well known that temperature can directly affect organisms exposed to these chemicals. Their metabolic rate, locomotion or feeding activity may be influenced by temperature thus affecting toxicant uptake and elimination as well as detoxification rates (e.g., Donker et al., 1998 and Garcia et al., 2011). In addition, temperature increase or decrease, also influences the fate of pesticides, mainly by altering their metabolization and degradation (e.g. Racke et al., 1997; Laabs et al., 2000 and Paraiba et al., 2003). Therefore, already about 20 years ago it has been proposed to adapt testing requirements to regional environmental conditions (Wiktelius et al.,
Corresponding author.
http://dx.doi.org/10.1016/j.ecoenv.2017.02.046 Received 5 September 2016; Received in revised form 25 February 2017; Accepted 27 February 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.
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Development, 2008, 2009). It contained 74.8% quartz, 20% kaolin clay, 5.2% sphagnum peat by dry weight and has a pH of 5.75 ± −0.5. The mixture was moistened to a maximum water holding capacity (WHC) of 60%.
1999). In the context of climate change, rising temperatures will even increase this problem (Noyes et al., 2009). The average global temperature is expected to increase by 1.4–5.8 °C in 2100 and this rise is expected to be more in the Temperate regions (FAO Food and Agriculture Organization, and IPCC, 2002, 2014). Few studies have investigated the effect of temperature on pesticide toxicity to soil organisms. For example, Garcia (2004) assessed the influence of temperature on toxicity of two fungicides (carbendazim and benomyl) and an insecticide (lambda-cyhalothrin) on the earthworm Eisenia fetida and the isopod Porcellionides pruinosus. He found lower toxicity for the fungicides and higher toxicity for the insecticide at tropical (28 °C) compared to temperate (20 °C) conditions. De Silva et al. (2009) studied the effects of temperature (20 vs 26 °C) on the toxicity of organophosphate pesticides to the earthworm E. andrei and found that toxicity was higher at higher temperatures. Recently, Bandow et al. (2014) observed increased susceptibility of the Collembolan Folsomia candida to a fungicide, pyrimethanil at a temperature of 26 °C compared to 20°C. Even interactions between effects of the insecticide lambda-cyhalothrin and soil moisture could be identified (Bandow et al., 2014). However, information on the toxicity of pesticides at different temperatures to soil organisms is still limited at best. For example, the influence of temperature on the pesticide toxicity to predatory mites has not been tested at all. Therefore, studies on the effect of pesticides on soil organisms at increased temperatures are necessary to correctly quantify their risk in the tropics. Insecticides are commonly used pesticides in many tropical regions and by design, are very potent in affecting different invertebrate species (Heong and Schoenly, 1998). Micro-arthropods (i.e. Acari and Collembola) are important inhabitants of agricultural soils, either as pests, as pest antagonists or as decomposers (i.e. influencing nutrient cycling via regulating microbial communities). These animals tend to be directly exposed to insecticide used in farmlands. The objectives of this study is to assess, the effects of temperature on the toxicity of three commonly and globally used insecticides,; a pyrethroid (deltamethrin) and two organophosphates (chlorpyrifos and dimethoate) on two standard micro-arthropods; Folsomia candida; a collembolan and Hypoaspis aculeifer, a gamasid mite.
2.3. Test substances Test chemicals used included three pesticides: dimethoate (an organophosphate, added as the formulation Bi 58, containing 400 g a.s./L, Compo GmbH, Germany) and chlorpyrifos (an organophosphate, added as the formulation Pestanal, purity: 99.9% CAS No: 2921-88-2, Sigma-Aldrich, Germany) and deltamethrin (a pyrethroid, added as the formulation Decisflussig, 2.8% active substance (a.s), Bayer Crop Science GmbH, Germany). The concentrations used in the individual tests were based on the results of range-finding-tests, covering a range between 0.1 and 1000 mg a.i./kg dw. 2.4. Test procedures Each treatment including the control had four replicates. Dimethoate and deltamethrin were added to the soil as aqueous solution in an amount of de-ionized water in order to reach 50% of the maximum WHC of the soil. For chlorpyrifos, the compounds were introduced to a portion (10%) of the soil, using acetone (1:2, v/w soil) as a carrier, and left in a fume hood for at least 24 h to allow the acetone solvent to evaporate after which it was mixed with remaining portion (90% of total soil). The soil was then subsequently moistened to the required moisture level of 50% of the maximum WHC. Solvent controls were also prepared for the test. Prepared soil treatments were introduced into the test vessels and the animals were introduced immediately afterwards. The mite test was performed as described in OECD Organization for Economic Co-operation and Development (2008). Ten adult female mites between ages 28–35 days were introduced in each test vessel containing 20 g dry weight soil. Each test vessel was covered with a paraffin film which was perforated to allow gaseous exchange. All test vessels were kept in climate chambers at 20 °C and 28 °C for 14 days and the test organisms were fed every 3 days with cheese mites. The temperature range was fixed based on information regarding the ecological preferences of this species (Jänsch et al., 2005). Moisture changes were assessed weekly by weighing the vessels and the corresponding water loss was replenished. After the 14th day of test, the test vessels were removed from the climate chamber and the mites were extracted using a modified Berlese-Tullgren apparatus. The extraction took about 2 days where the soils were exposed to light at temperatures of 25 °C for 12 h, 35 °C for 12 h and 45 °C for 24 h to ensure total recovery of the mites (OECD Organization for Economic Co-operation and Development, 2008). The Collembolan test was performed as described in OECD Organization for Economic Co-operation and Development (2009). Ten juvenile organisms (10–12 days) were introduced into glass vessels (50 ml), each containing 20 g of dry soil which had been mixed thoroughly with de-ionized water (controls) or the chemicals. The test vessels were kept in climate chambers at 20 °C and 26 °C for 28 days and food (5 mg of dry yeast) was added twice per week to each container. The temperature range was fixed based on information regarding the ecological preferences of this species (Jänsch et al., 2005). After 28 days, the test was ended. Each test vessel was emptied into a container filled with distilled water for extraction. The collembolans floated on the water surface. The adults and juveniles were counted separately in order to determine survival and reproduction. In order to facilitate the counting of the springtails, a black ink was added to the water to give a black background color against the white colors of the collembolans. The collembolans were counted under a light microscope.
2. Materials and methods 2.1. Test species The predatory mite Hypoaspis (Geolaelaps) aculeifer Canestrini (Acari: Laelapidae) was used in this study. The mites were taken from a culture bred in the laboratory of ECT Oekotoxikologie GmbH, Floersheim, Germany. They were kept at 20 °C with a photoperiod of 16:8 h, light: dark, in plastic containers lined with an 8:1 ratio of Plaster of Paris and charcoal. The substrate was moistened once a week with de-ionized water, and cheese mites (Tyrophagus putrescentiae) were added ad-libitum as a food source. Specimens used for the test were adult, 28–35 days old and were collected from a synchronised culture. The other test species was the springtail Folsomia candida (Collembola: Isotomidae). A culture of F. candida has been maintained for more than ten years at the laboratory of ECT Oekotoxikologie GmbH, Floersheim, Germany. They were reared in plastic containers lined with a medium of an 8:1 ratio of Plaster of Paris and charcoal maintained at 20 °C with a photoperiod of 16:8 h, light: dark, which was moistened weekly. A small amount of bread yeast was added ad libitum. Juvenile collembolans aged 10–12 days were used in the tests. 2.2. Test soil Artificial soil prepared according to the guidelines of the Organization for Economic Co-operation and Development was used in the experiments (OECD Organization for Economic Co-operation and Development, and OECD Organization for Economic Co-operation and 215
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3. Data assessment
In the tests with dimethoate (Fig. 1), at 28 °C percentage survival was higher than 100% in the control and also at 1.00 mg a.i./kg dw (not at 1.78 mg a.i./kg dw). The reason was that one could not differentiate the surviving adults from some rapidly maturing juveniles. Other juveniles in this case were differentiated by their lighter brown color but no juvenile was white as it is supposed to be. At 28 °C, no mites survived at the two higher concentrations, which happened at 20 °C only at the highest concentration. In general, survival was clearly lower at 28 °C than at 20 °C, as indicated by the LC50 values which do not overlap (Table 2). Effects of dimethoate on mite reproduction were similar at both temperatures (see EC50/EC 10 values; Table 2), but the number of juveniles was much lower at all concentrations at 28 °C compared to 20 °C. At 28 °C no juveniles were found starting from 5.62 mg/kg concentration and a strong decline in reproduction was found for the same concentration at 20 °C. For chlorpyrifos, at both temperatures very few mites survived at the highest concentration of 10 mg a.i./kg dw (Fig. 1). The higher toxicity of chlorpyrifos on survival at 28 °C than 20 °C is indicated by the LC50 values which differ by a factor of about two (Table 2). At 28 °C, significant effects on reproduction started at 0.32 mg a.i./kg dw, whereas at 20 °C significant effects (ANOVA, p < 0.05) on reproduction were found quite abruptly only at 10 mg a.i./kg dw(Fig. 1). Again, absolute juvenile numbers at all concentrations were much lower at the higher temperature. The EC50 values did not differ much from the LC50 values at the respective temperature, but they were by a factor of about four lower at 28 °C than at 20 °C (Table 2). For deltamethrin, there was no significant effect (p > 0.05) on survival up till 10 mg a.i./kg dwat both 28 °C and 20 °C (Fig. 1). At both temperatures, only very few individuals survived at 32 mg a.i./kg dw; i.e. there was a sharp increase in toxicity. At 28 °C, survival was higher than 100% in the control and two concentrations (1 and 3.2 mg a.i./kg dw, but at 0.32 mg a.i./kg dw the opposite was found), again indicating quicker development at the higher temperature. The LC50 values did not differ at the two temperatures (Table 2). At 20 °C, juvenile numbers did not differ between the control and 3.2 mg a.i./kg dw, but decreased by ca. 50% at 10 mg a.i./kg dw and were zero at the highest concentration of 32 mg a.i./kg dw. At 28 °C° toxic effects on mite reproduction decreased at an (almost) constant rate over the whole concentration range. Consequently, the EC50 values were by a factor of four higher at 20 °C than at 28 °C – and the EC10 values differed even by a factor of 30 (Table 2). In summary, out of the nine comparisons (three endpoints and three pesticides) which are shown here, toxicity increased with increasing temperature in six cases (Higher sensitivity at 28 °C. LC50 of dimethoate, chlorpyrifos, EC50 of chlorpyrifos and deltamethrin, EC10 of chlorpyrifos and deltamethrin.). In the other three cases (Lower sensitivity at 28 °C LC50 of deltamethrin, EC50 of dimethoate and EC10 of dimethoate.), there was no difference in the LC/EC values based on the observation whether there was an overlap of the confidence intervals or not. Not surprisingly, this overlap was most pronounced for the EC10 values, i.e. in all three cases. There is no clear correlation between the test chemical and the toxicity pattern in relation to the change in temperature.
3.1. Statistics Nominal concentrations of pesticides were used for calculations. The LC50 values were calculated using mortality data and the Trimmed Spearman-Kärber Program version 1.5, because a good fit was not obtained with probit analysis. The EC50and EC10 values of the pesticides for reproduction were calculated using the Linear Interpolation Method (USEPA, 1993) or non-linear regression methods (logistic 3-parameter) methods respectively, depending on which model gave a higher adjusted r2 value. In cases where the Linear Interpolation method was adopted for analysis, the expanded confidence limits were used because the number of replicates per treatment was less than seven (Environment Canada, 2005). Differences between the reproduction EC50 values on one hand and the LC50 values on the other hand for the two temperature regimes were ascertained when confidence limits did not overlap. The effect of increasing pesticide concentration on survival and reproduction of the collembolans and mites was assessed with a one way analysis of variance (ANOVA). The tukey post-hoc comparison was used to check where the differences lied. To test the direct effect of temperature on survival and reproduction of the mites and collembolans in control treatments, student's t-test was used. 3.2. Preliminary risk assessment of pesticides at tropical temperatures A preliminary risk assessment of the three pesticides was carried out by estimating the risk using the Toxicity Exposure Ratio (TER) as described by Römbke et al. (2008), i.e. following the rules used for the registration of pesticides in the European Union (EC (European Commission) and EC (European Commission), 2002, 2009). For the acute risk, the TERacute is the ratio of the LC50 to the predicted environmental concentration (PEC). For all pesticides and soils a single application and the highest normal application rate (deltamethrin: 224 g/ha (EFSA (European Food Safety Authority), 2010); dimethoate: 748 g/ha (EFSA (European Food Safety Authority), 2013); chlorpyrifos: 648 g/ha (EFSA (European Food Safety Authority), 2014) was used when calculating the PEC (based on a soil depth of 5 cm and a soil bulk density of 1.5) (Table 1). If the TERacute ≥10, a low acute risk is indicated. For the chronic risk, the TERchronic is the ratio of the NOEC or EC10 to the PEC. The EC10 was used instead of the NOEC because it is relatively more precise (Warne and van Dam, 2008). If the TERchronic > 5, a low chronic risk is indicated. 4. Results 4.1. Survival and reproduction of Hypoaspis aculeifer In the control vessels, validity criteria (≥80% survival) and (≤30% coefficient of variation) were met in all survival tests (OECD Organization for Economic Co-operation and Development, 2008). In general, the number of juveniles produced in the controls was significantly lower (t-test, p < 0.05) at 28 °C than at 20 °C.
Table 1 Calculation of the predicted environmental concentration (PEC) for the three pesticides used (all concentrations refer to dry weight).
Soil depth (d) Bulk density (D) Vol (V)=d x length (l) x breadth (b) Mass (M)=D X V Application rate (a.r) a.r converted to g/m2(1 ha = 10,000 m2) PEC in mg/kg (Given 1 m2=75 kg of soil) PEC
Dimethoate
Chlorpyrifos
Deltamethrin
0.05 m 1500 kg/m3 0.05×1×1=0.05 m3 0.05×1500=75 kg 748 g/ha 748/10,000=0.0748 g/m2 0.0748/75=1.00 mg/kg 1.00 mg/kg
0.05 m 1500 kg/m3 0.05 × 1×1 = 0.05 m3 0.05×1500=75 kg 648 g/ha 648/10,000=0.0648 g/m2 0.0648/75=0.86 mg/kg 0.86 mg/kg
0.05 m 1500 kg/m3 0.05×1×1=0.05 m3 0.05×1500=75 kg 224 g/ha 224/10,000=0.0224 g/m2 0.0224/75=0.30 mg/kg 0.30 mg/kg
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Fig. 1. Mean ( ± SE, n=4) percentage survival (left) and number of juveniles (right) produced by the predatory mite Hypoaspis aculeifer exposed to dimethoate, chlorpyrifos and deltamethrin in OECD soil under stable laboratory conditions at 20 °C and 28 °C. Surv 20= Survival at 20 °C, Surv 28= Survival at 28 °C, Repro 20= Reproduction at 20 °C, Repro 28= Reproduction at 28 °C.
nificantly between the two temperatures, but were less than a factor of two lower than the EC50 values. For chlorpyrifos, comparable effect patterns on reproduction as for dimethoate were observed at both temperatures (Fig. 2). The LC50 and EC50 were similar at both temperatures, but in absolute terms at very low concentrations (Table 3). Significant effects (ANOVA, p < 0.05) on reproduction were found at even the lowest concentrations tested, with very few or no springtails observed at the two highest concentrations (0.05 and 0.1 mg a.i/kg dw). This reflected in the EC50 and EC10 values because they were all very low (Table 3). For deltamethrin, toxic effects were lower at 26 °C than at 20 °C which was evident in the percentage survival recorded at both temperatures. This fact is also visible in the LC50 values which were by a factor of two higher at 26 °C (Table 3). This pattern of toxicity was also observed for reproduction, i.e. toxic effects of deltamethrin were lower at 26 °C than at 20 °C (Fig. 2). Consequently, this fact is also reflected by the EC50 at 26 °C (higher by a factor of four) and EC10 at 26 °C (higher even by a factor ten) values. In the tests with dimethoate toxicity always increased. In the tests with chlorpyrifos, toxicity based on survival was similar at both
4.2. Survival and reproduction of Folsomia candida The validity criteria (> 80% survival, > 100 juveniles, < 30% coefficient of variation) for Collembola were met for all tests (OECD Organization for Economic Co-operation and Development, 2008). Generally, there was no significant difference (p < 0.05) in the % survival at 26 and 20 °C in the controls but the number of juveniles produced in the controls was significantly lower (p < 0.05) at 26 °C than at 20 °C, with one exception: in the tests with chlorpyrifos the number of juveniles produced at 26 °C was generally higher than the ones produced at 20 °C but the difference was not statistically significant (p > 0.05). At both temperatures, effects of dimethoate on the survival of F. candida became significant from 0.4 mg a.i./kg dw (Fig. 2). With the exception of few individuals at 0.4 mg ai.i/kg dw and 20 °C no springtails survived at higher concentrations from 0.4 mg a.i/kg dw. Toxic effects on the survival of the collembolans were higher at 26 °C than at 20 °C as confirmed by the LC50 values, which differed by a factor of two (Table 3). At both temperatures the EC50 values were only slightly lower than the LC50 values. The EC10 values differed sig-
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Table 2 Comparison of the median lethal concentration (LC50) values (a), the median effective concentration (EC50) values (b), and the 10% effective concentration (EC10) values (c) with confidence intervals for the effects on survival and reproduction of the Predatory mite H. aculeifer exposed to dimethoate, chlorpyrifos and deltamethrin in OECD artificial soil at 20 °C and 28 °C.
Table 3 A comparison of the median lethal concentration (LC50) values (a) median effective concentration (EC50) values (b) and 10% effective concentration (EC10) values with confidence interval for the effects on survival and reproduction of F. candida exposed to dimethoate, chlorpyrifos and deltamethrin in OECD artificial soil at 20 °C and 26 °C. Pesticides
Pesticides
20 oC a) LC50 Dimethoate(mg/kgdw) Chlorpyrifos(mg/kgdw) Deltamethrin(mg/kgdw) b) EC50 Dimethoate(mg/kgdw) Chlorpyrifos(mg/kgdw) Deltamethrin(mg/kgdw) c) EC10 Dimethoate(mg/kgdw) Chlorpyrifos(mg/kgdw) Deltamethrin(mg/kgdw)
Temperature
Temperature
4.60 (3.90 − 5.00) 5.50 (5.10 − 5.80) 16.30 (13.60 − 19.40)
4.00 (3.55 − 4.45) 6.18 (−9.94 − 22.30) 10.09 (9.11 − 11.07)
2.69 (2.17 − 3.20) 5.09 (−11.05 − 21.24) 7.51 (−8.08 − 23.11)
20 °C
26 °C
a) LC50 Dimethoate(mg/kgdw) Chlorpyrifos(mg/kgdw) Deltamethrin(mg/kgdw)
0.36 (0.32 −0.40) 0.04 (0.03–0.05) 6.84 (5.93–7.89)
0.17 (0.15 − 0.19) 0.04 (0.03–0.04) 11.27 (8.52–14.90)
b) EC50 Dimethoate(mg/kgdw) Chlorpyrifos(mg/kgdw) Deltamethrin(mg/kgdw)
0.29 (0.26 − 0.33) 0.031 (0.023 − 0.038) 2.77 (1.04 − 4.49)
0.11 (0.09 − 0.13) 0.018 (0.01 − 0.024) 12.85 (7.72 − 17.98)
c) EC10 Dimethoate(mg/kgdw) Chlorpyrifos(mg/kgdw) Deltamethrin(mg/kgdw)
0.21 (0.17 − 0.25) 0.016 (0.005 − 0.26) 0.57 (−0.29 − 1.43)
0.055 (0.035 − 0.075) 0.005 (0.001 − 0.009) 5.40 (−2.26 − 13.07)
28 oC
2.82 (2.50 – 3.10) 2.41 (1.7 – 3.31) 17.50 (16.40 – 18.70)
3.39 (0.86 − 5.92) 1.42 (0.94 − 1.91) 2.52 (0.89 − 4.17)
2.90 (0.34 − 5.46) 0.71 (0.23 − 1.20) 0.24 (0.1 − 0.59)
Fig. 2. Mean ( ± SE, n=4) percentage survival (Left) and number of juveniles (right) produced by the collembolan Folsomia candida exposed to dimethoate, chlorpyrifos and deltamethrin in OECD soil under stable laboratory conditions at 20 °C and 26 °C. Surv 20= Survival at 20 °C, Surv 26= Survival at 26 °C, Repro 20= Reproduction at 20 °C, Repro 26= Reproduction at 26 °C.
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Table 4 Risk assessment of dimethoate, chlorpyrifos and deltamethrin at tropical temperatures(28 °C/26 °C), based on the comparison of acute (LC50) and chronic (EC10) effect values determined in laboratory tests with two micro-arthropods with predicted environmental concentrations (PECs) based on recommended application rates in agriculture (always one application of the respective pesticide). A risk is indicated (* and bold) when the Toxicity Exposure Ratio (TER) values are below ≤10 (acute tests) or ≤5 (chronic tests) (EC (European Commission), 2002). All concentrations are given as mg ai/kg dw. a. Hypoaspis aculeifer Pesticide
Rate a.i. (g/ha)
PEC (mg/kg)
20 °C LC50
20 °C Acute TER
28 °C LC50
28 °C Acute TER
20 °C EC10
20 °C Chronic TER
28 °C EC10
28 °C Chronic TER
Dimethoate Chlorpyrifos Deltamethrin
748 648 224
1.00 0.86 0.30
4.60 5.50 16.30
4.60* 6.40* 54.33
2.80 2.40 17.50
2.80* 2.79* 58.33
2.69 5.09 7.51
2.69* 5.92 25.23
2.90 0.71 0.24
2.90* 0.83* 0.80*
b. Folsomia candida Pesticide
Rate a.i. (g/ha)
PEC (mg/kg)
20oCLC50
20oC Acute TER
26oC LC50
26oC Acute TER
20oC EC10
20oC Chronic TER
26oC EC10
26oC Chronic TER
Dimethoate Chlorpyrifos Deltamethrin
748 648 224
1.00 0.86 0.30
0.36 0.04 6.84
0.36* 0.05* 22.80
0.17 0.04 11.27
0.17* 0.05* 37.57
0.21 0.016 0.57
0.21* 0.019* 1.90*
0.055 0.005 5.40
0.055* 0.006* 18.00
Acute TER = LC50/PEC; Chronic TER = EC10/PEC.a.i (g/ha) = active ingredient in gram/hectare
that these tests were performed slightly out of the range of preference of H. aculeifer while still be in the range of tolerance (Jänsch et al., 2005). However, since the results even in these two tests with relatively low number of juveniles did follow a dose-response-curve and other validity criteria such as the required range of variation were fulfilled we consider the data as relevant for our study. In future tests and referring to the information gained in our tests, it might be better to perform mite tests mimicking tropical conditions also at 26 °C (see also next paragraph). A temperature of 28 °C representative of tropical temperatures against a temperate temperature of 20 °C was initially chosen for the tests with springtails (F. candida) too. However, according to information collected by Jänsch et al. (2005) a temperature of 26 °C is likely the upper limit of the range of tolerance for F. candida. Therefore, the temperature for the collembolan tests was lowered down to 26 °C. Since 26 °C is still within the range of tropical temperatures generally known to be within 25–35 °C, the results of this study could still be considered to be relevant for tropical environment.
temperatures but toxicity based on reproduction increased at 28 °C than at 20 °C. Deltamethrin caused either no difference in toxicity (EC50) or toxicity even decreased with increasing temperature. In summary, out of the nine comparisons (three endpoints and three pesticides) which are shown here, toxicity increased with increasing temperature in five cases, in three cases toxicity decreased and in one case there was no difference in toxicity. 4.3. Preliminary risk assessment of pesticides at tropical temperatures Referring to the rules of the European Union for the risk assessment of pesticides (EC (European Commission) (2002)), a risk for soil microarthropods cannot be excluded when comparing recommended application rates with effects determined in laboratory tests with the species H. aculeifer and F. candida (Tables 4a & 4b). In the tests with the mite H. aculeifer and dimethoate an acute and chronic risk is indicated at both temperatures. Almost the same result was found in the tests with chlorpyrifos, with one exception: no risk was found when comparing the chronic test results gained at 20 °C with the PEC. In the tests with deltamethrin a risk was found only in the chronic test at 28 °C. In summary, for the three insecticides clearly different outcomes in the risk assessment procedure were determined and a risk was found more often at 28 °C (five times) than at 20 °C (three times). In the tests with the springtail F. candida and dimethoate acute and chronic risks are also indicated at both temperatures. The same outcome was found in the tests with chlorpyrifos. In contrast to the other species, however, in the tests with deltamethrin a risk was only indicated at the lower temperature (20 °C). Thus, this species reacts slightly more sensitive than the mites – and also slightly more often at the lower (five times) than the higher (four times) temperature.
5.2. Interaction of temperature and pesticides on toxicity to microarthropods This study shows that temperature influenced the toxicity of the three tested pesticides to the two micro-arthropod species (H. aculeifer and F. candida). The organophosphates dimethoate and chlorpyrifos were highly toxic to both species at both temperatures. In fact, F. candida was clearly more sensitive than H. aculeifer (with one exception: the test with deltamethrin at 28 °C) – a relationship which has been found already for many other chemicals (Huguier et al., 2015). In contrast, the third insecticide, the pyrethroid deltamethrin, was less toxic (especially for F. candida). Interestingly, this difference is not very pronounced at 20 °C, but clearly at 26 °C. It might be that the concentration of deltamethrin decreased more quickly at the higher temperature, since in the literature the mean degradation time (DT50) under temperate conditions is given as 35 days, while in Brazilian field sites a much lower DT50 value of about 12.4 days has been reported at higher, but not specified temperatures (Laabs, and Römbke et al., 2002, 2008). However, physiological reasons may also play a role whereby collembolans possibly were able to metabolize and eliminate deltamethrin faster at high temperatures. Looking at the absolute toxicity values, it seems that, independent from the endpoint (mortality, reproduction), toxicity increased by a factor of two to four with increasing temperature. However, these differences were not always significant because of the variability of the
5. Discussion 5.1. Temperature effect on microarthropods Usually, tests with mites and springtails are performed according to standard methods (OECD Organization for Economic Co-operation and Development, 2008, 2009), i.e. at a temperature of 20 ± 2 °C. We could show that the same tests with the same species can also be performed at 28 °C (mites) and 26 °C (springtails) – and with the exception of two mite reproduction tests in which one validity criterion was not met (i.e. less than 100 juveniles were found in the controls) all of them were valid. In the light of the fact that juvenile numbers were always lower in all tests running at 28 °C compared to those performed at 20 °C it seems 219
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more frequently (Joose and Buker, 1979). The difference in response of F. candida and H. aculeifer to deltamethrin at high and low temperatures could only be attributed to species differences. The preliminary risk assessment showed that elevated temperature, typical of tropical areas, could enhance the risk of these pesticides to mites and collembolans in the soil. However, the differences in toxicity were often not high enough in order to increase the risk considerably. In addition, these insecticides, might not be the best substances for studying this effect, because they are already very toxic at lower temperatures such as 20 °C. In addition, one may ask whether the risk classes (i.e. the trigger values of 10 and 5) are appropriate. However, in any case it is unlikely that higher temperatures cause a decrease in toxicity and, thus, risk – this happened only twice in the tests with F. candida – in all other cases toxicity and risk either stayed similar or increased. There is another factor to be considered in this discussion: In the present study, just one application was used to calculate the PEC. However, most of the time, farmers in the developing tropical countries spray their farmlands more than once per season, independently from respective application rules. In such cases, the PEC will increase, meaning that the risks described here will increase. Already now, a high risk may occur when using the two organophosphates – and also deltamethrin cause a risk at higher temperatures (at least for mites).
individual endpoints. This pattern was observed for the OP pesticides (dimethoate and chlorpyrifos) for both species and for the pyrethroid (deltamethrin) in tests with H.aculeifer. The toxicity of deltamethrin at high temperature did not follow a specific pattern; both increases and decreases could be observed depending on the species. Some studies (Weston, and Harwood et al., 2009, 2009) have shown that pyrethroids tend to be more toxic to organisms at lower temperatures. For example, there was a 2–13 fold increase in toxicity to cockroaches, tobacco budworms, cabbage looper, potato beetles at about 10 °C decrease in temperature (Harwood et al., 2009). Pyrethroids are sodium channel modulators and as such, at reduced temperatures, there is an increased stability of open-modified sodium channels which further prolongs the duration of sodium influx, thereby increasing the vulnerability of the nervous system to toxic effect. This trend was demonstrated in our study with F. candida but it is not exactly clear why this trend was not found for the mite H. aculeifer in this study. More generally speaking, at higher temperatures more or other metabolites could occur, meaning uptake, distribution, transport and metabolization of chemicals within the organisms might be changed (Howe et al., 1994), processes which are also discussed in the context of climate change (Noyes et al., 2009). However, for the scope of this study, we wanted to investigate toxicity to mites and collembolans at high and low temperatures and not specifically on the mechanism of toxicity. In summary the outcome of some of these tests was not really expected, since referring to the literature, effects should increase at higher temperatures. For example, in a recent study (Bandow et al., 2014), increasing toxicity of the fungicide pyrimethanil to the collembolan F. candida was observed at a temperature of 26 °C when compared to the results gained at 20 °C. However, this study shows that toxicity may be species or chemical specific. A study such as this with tropical species could have given more information. But according to the review of Huguier et al. (2015), no such studies were performed with tropical mites so far. The only exception refers to a moss mite, Archegozetes longisetosus (Oribatida; Trhypochthoniidae), which was tested with cadmium at 30 °C (Seniczak et al., 2009). So, studies on the effects of pesticides to other arthropods were also reviewed as well as experiences with other invertebrates exposed to our test chemicals. Garcia (2004) observed increased toxicity of the pyrethroid insecticide lambda-cyhalothrin to the isopod Porcellionides pruinosus (and the earthworm Eisenia fetida) at 28 °C when compared to 20 °C. This finding was supported by De Silva et al. (2009) who reported increased toxicity of the organophosphate pesticide chlorpyrifos to the earthworm Eisenia andrei at 26 °C than at 20 °C. Römbke et al. (2008) observed that the pyrethroids deltamethrin and lambda-cyhalothrin did not pose risk to earthworms under tropical conditions in Central Amazonian soils, in contrast to the organophosphate parathion methyl. In that study, the same EU approach was used to assess the risk of pesticides, but these authors incorporated the median disappearance time (DT50) as well as the number of pesticide applications within one season. However, this kind of data can only be gathered site-specifically, i.e. we could not include these refinements in our study. Our results here are supported by literature data, indicating the same trends of increasing toxicity at increasing temperatures. Because only few studies of this kind had been performed with H. aculeifer and F. candida so far, we also compared the results of our studies with those performed with other soil invertebrates which also could be exposed to pesticides. The other pattern of toxicity was reduced toxicity of pesticides at increased temperature, as found for deltamethrin on F. candida in which toxic effect on survival and reproduction of F. candida was higher at 20 °C than at 26 °C by almost by a factor of 2. This observation could be due to degradation of deltamethrin which is faster at higher temperatures of 25 °C than at 20 °C in the laboratory under standardized conditions (Bayer Crop Science, 2009). Also, at high temperatures, excretion of toxic substances by molting and intestine renewal occur
6. Conclusions The study has shown that changes in temperature can influence the toxicity of chlorpyrifos, dimethoate and deltamethrin to soil microarthropods and re-emphasize that temperature is an important abiotic factor which is necessary for proper soil functioning. The study also showed that species and pesticide differences could influence the toxic response when temperature changes. As in the case of Folsomia candida, it was less sensitive to deltamethrin at high temperatures compared to Hypoaspis aculeifer which was sensitive to deltamethrin at higher temperature. Based on the data from this study where tests were carried out at temperate (20 °C) and tropical temperatures (26 and 28 °C), it could be deduced that toxicity of pesticides are generally higher under tropical conditions. For the purpose of risk assessment, the toxicity were not high enough to increase risk remarkably. However, due to temperature differences asides other climatic conditions extrapolation of data from temperate climate to tropical climate should still be used with caution. It is also noteworthy to incorporate the disappearance time for the pesticides for future work and use a tropical soil for this kind of study as in Römbke et al. (2008). This study also provides some preliminary data that could show how previous risk assessment evaluations might be inaccurate in the context of global warming. Acknowledgments We would like to thank Adam Scheffczyk (ECT Oekotoxikologie GmbH) who was involved with helping to perform these tests. References Afari-Sefa, V., Asare-Bediako, E., Kenyon, L., Micah, J.A., 2015. Pesticide use practices and perceptions of vegetable farmers in the cocoa belts of the Ashanti and Western regions of Ghana. Adv. Crop Sci. Technol. 3, 174. http://dx.doi.org/10.4172/23298863.1000174. Bandow, C., Karau, N., Roembke, J., 2014. Interactive effects of pyrimethanil, soil moisture and temperature on Folsomia candida and Sinella curviseta (Collembola). Appl. Soil Ecol. 81, 22–29. Bayer Crop Science, 2009. In: Proceedings of the Pyrethroid Scientific Forum 2009 (2) Vol 62. De Silva, C.S.M., C.S., Van Gestel, C.A.M., 2009. Development of an alternative artificial soil for earthworm toxicity testing in tropical countries. Appl. Soil Ecol. 43, 170–174 (2009). Donker, M.H., Abdel-Lateif, H.M., Khalil, M.A., Bayoumi, B.M., Van Straalen, N.M., 1998. Temperature, physiological time, and zinc toxicity in the isopod Porcellio scaber. Environ. Toxicol. Chem. 17, 1558–1563.
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