Earthworms in Short-Term Contact with a Low Dose of Neonicotinoid Actara 25 WG

A laboratory experiment, carried out for five months, was designed to assess the effects produced by the neonicotinoid Actara 25 WG, used at a dose recommended by the manufacturer, in the dynamics of the populations of epigeic Eisenia fetida (Sav.) and Dendrobena veneta (Rosa)earthworms. The study was conducted in a climatic chamber and involved balanced biomass of mature specimens representing both species, in 3 replications of soil medium with the insecticide, in addition to controls. The growing populations were inspected five times, by means of manual segregation of the substrate. The insecticide led to an increase in both the number of specimens and the biomass of E. fetida population (overall, as well as mature and immature worms), yet it reduced their reproduction (decreased the number of cocoons) (p<0.05). In the case of D.veneta, the neonicotinoid insecticide produced positive effects only in the mature specimens (p<0.05).


INTRODUCTION
Due to the industrialization of agriculture and excessive use of chemicals, more and more toxic xenobiotics find their way into the natural environment.Approximately 13 million chemical compounds are already known and about 100,000 of these are manufactured in large quantities.The use of many of the chemicals leads to serious deterioration of the soil quality, partly resulting from the hazards towards the soil fauna.The latter must cope with xenobiotics and complex residues from their synergistic and antagonistic reactions (Walker et al. 2002;Laskowski & Migula 2004).
Chemical substances such as neonicotinoids are commonly used.It is estimated that this group of xenobiotics accounts for approximately 1/4 of all the insecticides manufactured globally (Jeschke et al. 2010, 2013; Wang et al. 2015a, b).They are widely used to eliminate harmful insects feeding on crops (e.g.sucking and chewing insects, such as whiteflies, thrips, aphids, certain microlepidoptera and beetles).Irrespective of the way they are absorbed by plants, neonicotinoids have a capacity to translocate to all parts of the organism.Therefore, they are able to control a large spectrum of pest insects (Simon-Delso et al. 2015).
Because of the wide range of systemic and translaminar effects and their unique activity, neonicotinoids rank among the most popular groups of insecticides.Yet, numerous researchers point out their negative influence on the functioning of ecosystems.This is partly associated with the non-target effects observed in the populations of pollinators (bees, bumblebees) and insectivorous birds.In the areas where these insecticides are used, related contaminations were identified in pollen range from 11 to 24% and in nectarfrom 17 to 65% (Kreutzweiser et al. 2009 Many studies suggest that even if they are used in the doses defined by their manufacturers as 'safe for the environment', xenobiotics are likely to affect numerous soil organisms, and consequences of their application cannot be predicted with sufficient certainty ( The experiment described here was designed to assess the effects of the insecticide Actara 25WG, on two species of earthworms.

Earthworms
The experiment used earthworms (Oligochaeta; Lumbricidae) from breeding stocks at the University of Rzeszów's Department of Natural Theories of Agriculture and Environmental Education.The experiment was designed to investigate two species: Eisenia fetida (Savigny 1826) and Dendrobena veneta (Rosa 1893), which were previously acclimatized in potting soil.

Experimental procedure
The study investigated balanced biomass of mature specimens of E. fetida and D. veneta.All specimens had a well-developed clitellum.Plastic containers (20×15×10 cm) were filled with potting soil and then 20 E. fetida or 10 D. veneta were placed in each of them (Table 1).In order to reduce the growth of potworms, competing with earthworms, kitchen waste subjected to vermicomposting was mixed with cellulose (in the ratio 2:1) (Kostecka 2000).The waste was supplied regularly, and placed in 5 mm mesh nylon net.
The experiment was conducted for 5 months (from December 2015 to May 2016).Every month the condition of the growing population of earthworms was inspected through the use of manual segregation of substrate (e.g.Pelosi et al. 2009).The experiment was carried out in a climatic chamber (20±5 o C, 24L; at soil moisture of approx.70%) (OECD 1984(OECD , 2004).

Statistical analysis
The findings are presented in the form of arithmetic means and standard deviations (SD).The statistical calculations were performed with the use of STATISTICA software v. 10 (StatSoft).Normal distribution was examined with Shapiro-Wilk W-test and Brown Forsythe test for verifying homogeneity of variances.Statistical significance was determined with Student's t-test a (Stanisz 2006).The significance of differences was assumed at α = 0.05.

Effects of insecticide Actara 25WG in E.fetida earthworms
The findings suggest positive effects of Actara 25WG (applied at the manufacturer's recommended dosage), in both the size and the biomass of mean population of E. fetida (mature and immature specimens) (size t=5.362, p<0.05; biomass t=32.47,p<0.001) (Fig. 1).
The positive response to the formulation of immature specimens was only observed in their number.Five months after the xenobiotic was applied, the boxes with the insecticide were found to contain 57±9 spec.·cont. - , compared to the controls amounting to 32±2 spec.·cont. - (t=4.6433,p<0.01).No differences were identified in the mean biomass of immature specimens (p>0.05)(Table 2).
The presence of the insecticide reduced the number of cocoons produced by earthworms (t=3.9043,p<0.05) without differentiating their biomass (p>0.05) (Fig. 3).

Effects of the insecticide Actara 25WG in D. veneta earthworms
Different response to the neonicotinoid insecticide was observed in D. veneta earthworms.The xenobiotic increased the size and biomass of mean populations of these worms, yet the effect was insignificant (size t=0.3207, p>0.05; biomass t=0.8562, p>0.05) (Fig. 4).
Actara 25WG only produced change in the number of mature D. veneta specimens.More of these were found in the containers with the xeno-   No effects of the formulation were identified in their biomass (p>0.05) (Fig. 5).The xenobiotic in question did not influence the population size (p>0.05)and biomass (p>0.05) of immature specimens (Table 3) or their reproduction (number of cocoons) (Fig. 6).
The findings show greater differences in the responses of specific specimens of D. veneta earthworm compared to E. fetida earthworm.Coefficients of variation in the relevant characteristics are presented in the following table (Table 4).

DISCUSSION
Neonicotinoid insecticides have been in use since the 1990s (Feltham et al. 2014).Their growing usage is associated, among others, with the wide range of their applications (they can be used in the form of both granulates and concentrates) (Sánchez-Bayo & Hyne 2014).The insecticides, however, present certain problems.These should be investigated not only because of the harmful impacts on honey bees (Sánchez-Bayo & Hyne 2014); it is also important to gain insight into the effects of exposing other organisms to these substances.There is still scarcity of evidence related to the impact of neonicotinoids on the soil environment, and more specifically earthworms, which are important representatives of soil ecosystems.They account for over 70% of biomass in soil and have a significant role in the functioning of the ecosystem (Blouin et al. 2013).Some authors, e.g.Sánchez-Bayo & Hyne (2014), argue that these insecticides constitute a real risk for earthworm populations, depending on the concentration of the medium and duration of exposure, as well as dosage and species-specific sensitivity.
Gomez   It has been established that when exposed to chemical stressors, earthworm may modify their energy balance and (depending on physiological  Since organisms have a specified amount of energy at their disposal, it is similar in a polluted and a so-called "pure" environment (Walker et al. 2002;Laskowski & Migula 2004).The use of energy from food is shown in Figure 7.It can be seen that when stressed, a specimen has to choose where to invest energy.The choice is linked with expending energy to detoxication (which should result in a greater chance of survival, yet it also reduces production) or with allocation of excess energy to production (which leads to a decrease in the level of detoxication).
The present study was conducted over a period of five months.It was designed to identify lifehistory parameters in the populations of E. fetida and D. veneta earthworms in the presence of the xenobiotic Actara 25 WG applied once.It was found that the insecticide produced varied effects in the mean populations of these earthworm species.This may be linked with a number of factors, including, firstly, the sensitivity of the two species.The differences in their sensitivity would be confirmed by large disparity in the coefficients of variation in the characteristics under consideration.The response of earthworms may also depend on the decomposition time of the chemical During the initial stage of the experiment involving E. fetida, a positive effect was observed in their response to low dose of the xenobiotic.As it was mentioned before, this was related to an increased production of cocoons, size (p<0.05)and biomass (p<0.001)not only in the entire population of earthworms but also to the population size (p<0.05)and biomass (p<0.001) of mature specimens, as well as population size of immature specimens (p<0.01).The application of the xenobiotic, however, affected the subsequent generation, as in the final stage of the experiment there was a decrease in the total number of cocoons produced by these earthworms (p<0.05).
As for D. veneta, the insecticide produced positive effects only in the mature specimens.At the end of the experiment, their number was significantly greater in the container with Actara 25WG (p<0.05).This response, observed in both annelid species, may also be explained by the phenomenon of hormesis, or the fact that an organism's defence system works in such a way as to enable beneficial effects, in response to small doses (Laskowski & Migula 2004).Furthermore, according to Calabrese (2005) the organisms exposed to low doses of chemicals frequently compensate for the negative impacts because such low doses may have stimulating effect, and at the next stage, the organism regains homeostasis.Alternatively, large doses frequently produce adverse effects, which are usually irreversible.
Alves et al. ( 2013) applied chronic toxicity tests to examine the influence of six insecticides (fipronil, imidacloprid, thiametoxam, captan, carboxin, and thiram) on Eisenia fetida andrei earthworms.Out of all these agents, only the neonicotinoid insecticide fipronil, applied at the lowest concentration (62.5 mg•kg -1 dry soil), produced a significant increase in the biomass of the exposed earthworms, compared with controls.Another neonicotinoid, i.e. imidacloprid, was found to significantly decrease the biomass.The remaining insecticides did not affect the red worms.
High acute toxicity of neonicotinoids (imidacloprid, clothianidin, nitenpyram, thiacloprid and acetamipryd) for mature E. fetida specimens was also reported by Wang et al. (2015a) who performed an experiment based on artificial soil test (OECD 1984).All the agents reduced the number and mass of cocoons, as well as the number of juveniles hatching from a single cocoon.The most toxic agent was clothianidin -even at the dose of 4.34 mg•kg -1 .
The negative effects of neonicotinoids were also confirmed by an earlier study conducted by Wang et al. (2012).Out of all the 24 xenobiotics, representing six classes of chemicals (acetamiprid, imidachlopryd, nitenpyram and thiacloprid, antibiotics, insect growth regulators IGR, pyrethroids, carbamates and organophosphates and others), their findings showed that neonicotinoids were most toxic for earthworms.These facts support the claim that neonicotinoids should be applied extremely cautiously or indeed their use should be restricted.

CONCLUSIONS
1.The findings suggest different sensitivity of E. fetida and D. veneta to the neonicotinoid Actara 25WG and provide evidence for varied life strategies in contact with stress caused by the agent.2. During a short survivability test (period of 5 months, 155 days), the insecticide applied at the dose recommended as safe by the manufacturer, produced varied responses in the populations of the two species of earthworms: -in E. fetida, low doses of Actara 25WG stimulated increase in the size (p<0.05)and biomass (p<0.001) of the mean populations, number of mature specimens (p<0.05) and their biomass (p<0.001) as well as the number of immature specimens (p<0.01).However, the application of Actara led to a reduced reproduction of E. fetida -there was a decrease in the number of cocoons produced (p<0.05),-in D. veneta, low dose of Actara 25WG only stimulated the size of mature populations (p<0.05).It did not affect the remaining characteristics (p>0.05).3. The research should be continued under varying conditions, because even though the findings suggest that partly successful life strategies are promoted, yet they also show reproductionrelated hazards for the investigated species of earthworms.

Figure 1 .
Figure 1.Effects of the xenobiotic Actara 25WG in the dynamics of size and biomass of mean population of earthworm E. fetida

1 Figure 2 .
Figure 2. Effects of the xenobiotic Actara 25WG in the dynamics of size and biomass of mature population of earthworms E. fetida

Figure 3 .
Figure 3. Effects of the xenobiotic Actara 25WG in the dynamics of size and biomass of cocoon population of earthworm E. fetida

Figure 4 .Figure 5 .
Figure 4. Effects of the xenobiotic Actara 25WG in the dynamics of size and biomass of D. veneta earthworm population

Figure 6 . 4 .*
Figure 6.Effects of the xenobiotic Actara 25WG in the dynamics of size and biomass of cocoon population of D. veneta earthworm

Figure 7 .
Figure 7. Model of standard use of energy obtained from food, based on a study of Eisenia fetida specimens (Johnston et al. 2014)

Table 2 .
Effects of xenobiotic Actara 25WG in the number and biomass of immature earthworms E. Fetida *-at the end of the month

Table 3 .
Effects of xenobiotic Actara 25WG in the number and biomass of immature D. Veneta earthworms Population size (specimen·container -1 ± SD)