Using earthworm avoidance behaviour to assess the toxicity of formulated herbicides and their active ingredients on natural soils

Background, aim, and scope Soil quality has been threatened by intensive agricultural practises, namely those relying on the application of pesticides, such as herbicides. Among the non-target terrestrial organisms exposed to such scenarios, earthworms are key ecological receptors widely used in ecotoxicological studies. As such, this work aims to assess the effects of two herbicide active ingredients (a.i.)--sulcotrione and penoxsulam--and their respective commercial formulations--MIKADO® and VIPER® (referred as Mikado and Viper)--on the avoidance behaviour of Eisenia andrei. In an attempt to enhance the ecological relevance of the generated toxicity data, the avoidance tests were run with standard (LUFA 2.2; L) and natural soils (from corn and rice fields), as long as their habitat function did not constrain the earthworm behaviour. Methodology Earthworms were bred in the lab before test conductance. The natural soils used as substrates were collected before the cropping season on corn (C) and rice (R) fields, which are integrated in a wide area exploited for agriculture. Their physico-chemical characterization evolved the determination of pH (H₂O, KCl), conductivity, organic matter (OM) and clay/silt contents, and water-holding capacity (WHC). The avoidance tests intended to ascertain (1) the random distribution of earthworms in the natural soils C and R (dual-control tests), (2) the habitat function of natural soils against each other and against L soil, (3) the effect of active ingredients and formulated herbicides on E. andrei behaviour. Avoidance tests with the a.i.s were only performed in L soil. Data evaluation followed ISO (2005) guidelines. Results C and R soils presented higher OM (5.1% and 4.5%, respectively) and clay/silt (53.3 and 43.1, respectively) contents and WHC (107.2 and 109.9%, respectively) than L soil (4.1, 21.4 and 48.0%, correspondingly). Earthworms distributed randomly in dual-control tests, but preferred R soil significantly, relative to L or C soils. The LOEC and EC₅₀ values calculated for sulcotrione (>1,000.0 and 1,263.3 mg a.i. kg⁻¹, respectively) and Mikado (1,012.8 and 1,301.3 mg a.i. kg⁻¹, respectively) were much higher than those calculated for penoxsulam (100 and 80.6 mg a.i. kg⁻¹, respectively) or Viper (52.7 and 51.5 mg a.i. kg⁻¹, respectively), when L soil was used as substrate. Moreover, the habitat function of L soil contaminated with the formulated herbicide Viper was more constrained relative to that of the a.i. penoxsulam. Viper induced higher % avoidance on E. andrei exposed to the contaminated L soil compared to that under the R soil. Discussion The response of earthworms to R (attraction) and C (avoidance) soils could be related, not only to the quantity of OM content, but also to the quality of organic and inorganic fractions of soil, beyond other intrinsic properties of soils. Both Mikado and sulcotrione impacted the behaviour of E. andrei only slightly. This endpoint was more affected under penoxsulam or Viper exposures on L soil, being the latter-formulated product even more repellent for E. andrei than the a.i. The effect of adjuvants added to the commercial formulation of Viper, may have increased the toxicity of the a.i. Thereby, our results reinforce the need for a careful assessment of the impacts of formulated products. Furthermore, since there was a reduction in earthworm % avoidance under Viper exposures on the natural soil R, it was possible that pesticide bioavailability had been reduced by its sorption to OM and clay mineral sorption sites. Conclusions Though the standard L soil should be used for reproducibility and comparison means, other natural soils should be added to the assessment of chemicals, for sake of ecological relevance. Both herbicides induced avoidance behaviour on E. andrei, albeit stronger effects were denoted by penoxsulam and its respective formulated product, Viper. Overall, avoidance tests provided a sensitive, valuable and feasible response either to compare the habitat function of different standard and agricultural natural soils or to test the effect of herbicides. Recommendations and perspectives An effort should be made to enlarge the terrestrial ecotoxicological database as a way to fulfil the huge lack of information available for this ecosystem. In this context, additional research congregating a potential linkage between physiological activities sustaining the regular metabolism of earthworms and their avoidance behaviour or even their reproductive effects would be welcomed, especially in what regards formulated pesticides. Such approach would provide a robust and comprehensive understanding of chemical effects. Furthermore, it is encouraged that natural soils should be used to improve the reliability of chemical testing.