Effect of herbicides on soil respiration: a case study conducted at Debrecen-Látókép Plant Cultivation Experimental Station

Measuring the effect of herbicides on the natural environment is essential to secure sustainable agriculture practices. Amount of carbon dioxide released by soil microorganisms (soil respiration) is one of the most important soil health indicators, known so far. In this paper we present a comprehensive quantifying study, in which we measured the effect of 14 herbicides on soil respiration over 16 years, from 1991 to 2017, at Debrecen-Látókép Plant Cultivation Experimental Station. Investigated herbicides contained different active ingredients and were applied in various doses. It was found that 11 out of the examined 14 herbicides had a detrimental effect on soil respiration.


Introduction
Carbon dioxide (CO 2 ) is an important greenhouse gas, which affects significantly global warming and climate change (Rastogi et al., 2002). Approximately 30% of the total CO 2 emissions are released by agricultural activities. It is notable that agricultural CO 2 emissions increased by 27% over two decades, from 1970decades, from to 1990decades, from (Lal, 2004. Primary sources of soil CO 2 emissions are root respiration and degrading of organics by soil microorganisms. Soil microbial activity mainly depends on soil properties, including soil temperature, organic matter and soil moisture content (Smith et al., 2003). Increasing scientific attention is focused on understanding the role of the soil microbial community (Bautista et al., 2017;Cho-Tiedje, 2000;Mátyás et al., 2018;Mátyás et al., 2020) and nutrient cycles (Jakab, 2020;Sándor et al., 2020). It has been documented that different cultivation technologies significantly impact soil microbiological activity (Sándor et al., 2020).
Different chemicals (such as fertilizers and/or herbicides) are utilized in agricultural technologies. Use of herbicides constitutes an integral part of crop production, and one should be aware that they cause a "secondary effect" on both soil life and so called "non-target" organisms (Kecskés, 1976). Sensitive organisms are killed after using herbicides, and their remains are easily decomposed by the surviving microorganisms (Cervelli et al., 1978). At present, the selection criteria for allowed chemicals is more rigorous and stricter than over past decades, and they are restricted to smaller concentrations (Inui et al., 2001). Soil microbes play a major role in maintaining soil quality (Mendes et al., 2018;Wang et al., 2008).
In this paper, we discuss carbon dioxide emission levels of chernozem soil at the Debrecen-Látókép Plant Cultivation Experimental Station, where herbicides were applied to control the weeds. We compare results of carbon dioxide production in treated plots to untreated control parcels.
Soil CO 2 was measured in triplicate by NaOH absorption. Experiments were performed between 1991 and 2017. In 1991In , 2000In , 2008 and 2017 soil samples were obtained two weeks after the herbicide(s) was applied. For incubation, 10 g of soil was weighed and placed in a polyester bag (0.1 mm ⌀ holes), from where CO 2 could escape. One took 500 mL laboratory glassware in which 10 cm 3 of 0.1 M NaOH solution (Sigma-Aldrich, USA) was introduced to absorb the released carbon dioxide. Soil samples were hung above the NaOH solution, and the glass containers were sealed tightly. Since CO 2 has a greater density than air, it sunk in the container, and was absorbed by the alkaline solution. After an incubation period of 7 days, the remaining alkali solution was back titrated with 0.1 M HCl (Sigma-Aldrich, USA), in the presence of phenolphthalein, and then with methyl orange indicator. From the volume of equivalence one can calculate the amount of CO 2 formed during soil respiration, according to Equation 1. mg (CO 2 )· 10 g −1 · 7 day −1 = (C-S) · f(NaOH) · f(HCl) · 2.2 * dm (1) where, C: 0.1 M/ dm 3 HCl loss for methyl orange indicator (Sigma-Aldrich, USA); S: 0.1 M dm 3 HCl loss for phenolphthalein indicator (Sigma-Aldrich, USA); f: 0.1 mol dm -3 HCl and a 0.10 mol dm -3 NaOH factor; 2.2: titer (1 mL 0,1 mol dm -3 HCl equivalent 2.2 mg CO 2 ); dm: multiplication factor for dry soil.
Data analysis was performed using Microsoft Excel 2003 (mean values and standard deviation). Two-factor variance analysis was performed to obtain the significant effect on measured parameters. Significant differences were accepted at the level 1%, but the evaluation was calculated by LSD 5% values, as widely accepted in agricultural research.

Results and discussion
In 1991, three herbicides were applied, and even the basic doses were high. Results were compared to the control; CO 2 production was significantly reduced at single doses and a further decrease was experienced at 2-3 times greater doses. Consequently, CO 2 production declined gradually with increasing doses of herbicides. The smallest production was obtained at 3 times the dose of Anelda Plus 80 EC, its value being only 59% of the control (Table 1).
In 2000, six different active ingredients were used, and their effect examined. Much lower doses were applied, half and one third of the ones used in 1991. As compared to the control, soil respiration decreased significantly in all treated plots, after laboratory incubation. The lowest results were obtained with Acenit 880 EC; when 3x dose was used, only the 64% of the control being achieved.
In 2008, a significant decrease was found for the treated soil relative to the control. In the treatment with triple dose, only 74% of the control was measured. The herbicides used in 2008 are no longer authorized, as they were withdrawn from the market.
In 2017, three herbicides were examined. Out of them, Figaro TF, which contained glyphosate agent, was no longer authorized. When this herbicide was applied, CO 2 production decreased significantly. Carbon dioxide production did not change considerably in Andengo and Capreno treatments; there was a slight increase in treatments with Andengo and decrease in treatments with Capreno.

Conclusions
We can conclude that CO 2 production decreased significantly in the soil for 11 out of the 14 herbicides. With two herbicides, Merlin SC (izoxaflutol) and Capreno (Isoxadifen-ethyl, tembotrione), there was no significant change of treated soils relative to the untreated soil, and there was only one herbicide Adengo (Bayer, Germany), which increased soil respiration slightly, but not significantly. The main sources of CO 2 -emissions from soil is the respiration of plant roots and of the microbial community. Therefore, a significant decrease of CO 2 emission indicates a change in these parameters. One can recommend for use those chemicals, which do not cause major changes in the microbial community and do not affect life conditions of other live organisms.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes respiration. In this study, they measured the effect of 14 herbicides on soil respiration over 16 years, from 1991 to 2017, at Debrecen-Látókép Plant Cultivation Experimental Station.This is an interesting and novel topics for the readers. There were some errors and shortcomings, which were following as: Q1: Title: In this title, it is no necessary to give the testing site. So it should be changed as "Effect of herbicides on soil respiration: a case study". ○ Q2: Abstract: Give more results of the measured indexes, not just the description of detrimental effects! Yes Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Soil microbiology
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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