Effects of the herbicide isoproturon on survival, growth rate, and protein content of mature earthworms (Lumbricus terrestris L.) and its fate in the soil

Applied Soil Ecology 23 (2003) 69–77

Effects of the herbicide isoproturon on survival, growth rate, and protein content of mature earthworms (Lumbricus terrestris L.) and its fate in the soil Yahia Y. Mosleh, Séverine Paris-Palacios, Michel Couderchet∗ , Guy Vernet Laboratoire d’Eco-Toxicologie, Faculté des Sciences, UPRES-EA 2069, Université de Reims Champagne-Ardenne (URCA), B.P. 1039, Reims 51687, France Received 10 July 2002; received in revised form 6 December 2002; accepted 11 December 2002

Abstract This study was conducted to investigate the effects of isoproturon on mature earthworms (Lumbricus terrestris L.) and its fate under laboratory conditions. Earthworms were exposed for various durations to soils contaminated with concentrations that were chosen to mimic an accidental spill of the herbicide. Residues were monitored in soil and earthworms after 7, 15, 30, 45, and 60 days of exposure to different isoproturon concentrations. Effects of the herbicide on mortality, relative growth rate, and total soluble protein content of earthworms were determined. No lethal effect of isoproturon was observed even at the highest concentration tested (1.4 g/kg soil) after 60 days of exposure. Residues of isoproturon caused a significant reduction in the growth rate (maximum −27.9%). Additionally a reduction in total soluble protein was observed in all treated worms (maximum −36.1%). The decrease in isoproturon concentration in the soil depended on initial concentration: it was slower at higher concentrations. In the worms, it increased during the first 15 days and decreased thereafter. It was concluded that an accidental spill of isoproturon may have localized consequences very different from those observed in cases of diffuse pollution, in terms of herbicide degradation and toxicity. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Lumbricidae; Phenyl urea; Pollution; Residue; Soil; Toxicology

1. Introduction The use of herbicides poses a risk of pollution of the subsoil and consequently of contamination of domestic water resources. Like other phenylurea herbicides, isoproturon is applied to soils, taken up by plant roots, rapidly transported through the plants, and inhibits photosynthesis in the leaves at the photosystem ∗ Corresponding author. Tel.: +33-326-91-3343; fax: +33-326-91-3342. E-mail address: [email protected] (M. Couderchet).

II level (Hock et al., 1995). Isoproturon is used for pre- and post-emergence control of monocot and dicot weeds in winter wheat. In Champagne, France, it is one of the most extensively used herbicides and, because of its properties, it belongs to the group of pesticides that are most likely to be recovered in underground and surface waters. Continuous application of isoproturon may lead to diffuse soil pollution, generating low concentrations of toxicants in the soil over large areas. In contrast, accidental spills or rinsing of spray equipment may result in high concentrations of herbicide on smaller areas. Evaluation of soil contamination has become a prior-

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ity for OECD countries (IICC, 1994). Earthworms are particularly important soil macroinvertebrates. Their burrows increase soil aeration and drainage. Moreover, the worms bring about a notable transportation of the lower soil to the surface. During these processes they improve soil structure. The amount of soil passing through their bodies annually can equal 6.3 t of dry earth per hectare (Edwards and Lofty, 1977). During passage through the worm not only organic matter, which serves as food, but also the mineral constituents of soil are subjected to digestive enzymes and to a grinding action within the animal body. Additionally, earthworms are known to accumulate heavy metals, pesticides, and other organic chemicals supporting their importance as a model for assessing the general impact of pollution on the soil community (Callahan, 1984). Governmental regulatory agencies utilize earthworms as biomonitors to determine the ecological hazards of heavy metal and pesticide-contaminated soils (OECD, 1984; Stürzenbaum et al., 1998). In addition, many biomarkers have been developed in earthworm species (Scott-Fordsmand and Weeks, 2000). Earthworm biomarkers have shown promising applications. These biomarkers include the decrease of protein content and enzyme activities in response to agrochemicals (Ismail et al., 1997a; Mosleh et al., 2003). The importance of earthworms in ecotoxicological studies is well recognized, but there is still a need for further data on the toxicity of specific pesticides to such non-target organisms in order to be able to select chemicals that cause little harm to them. This paper investigates effects of isoproturon concentrations, that may occur after an accidental spill, on the earthworm Lumbricus terrestris L. Since one of the earliest consequences of pesticide contamination on earthworms was found to be a decrease in protein content (Ismail et al., 1997b), the effect of isoproturon on protein content in the animal was assessed, together with mortality and growth. The fate of the herbicide in soil and earthworms is also reported.

that has not been treated with pesticides for 35 years. The worms were maintained in a soil mixture at 14 ± 1 ◦ C for 3 months before the experiments. The mixture was obtained by homogenizing soil samples (top 20–25 cm) taken from the area described above. Key physical and chemical properties of the soil mixture include: 28.7% sand, 27.4% silt, and 43.9% clay, 7.7% organic carbon, pH 8.16, and exchangeable P, K, Mg, and Na concentrations of 187, 288, 166, and 6 mg/kg, respectively. Earthworms used in this study were adults with a well-developed clitellum. They were removed from the soil 24 h before use and stored in Petri dishes on damp filter paper (in the dark at 14 ± 1 ◦ C) to void gut contents. 2.2. Toxicological tests Tests were conducted to determine the toxicity of isoproturon [N-(4-isopropylphenyl)N ,N -dimethylurea] for mature earthworms. The procedures for these experiments were based on those used by Heimbach (1984) as modified by Ahmed et al. (1991). Different quantities of isoproturon formulated as Matin® (Sipcam-Phyteurop, Levallois-Perret, France, suspension containing 500 g/l isoproturon) were dissolved in 100 ml of distilled water and mixed with the soil in 500 cm3 containers. Two containers were prepared for each sampling time and for each final concentration of isoproturon (0.5, 0.8, 1.0, 1.2, and 1.4 g/kg soil). Then, 10 mature earthworms were weighed and introduced into each container that was placed in an incubation chamber (14 ± 1 ◦ C, 70–90% relative humidity and 12:12 photoperiod). Two containers per sampling time received no isoproturon and were used as controls. After 7, 15, 30, 45, and 60 days the containers were sacrificed. The earthworms, they contained, were placed on damp filter paper to void gut contents, weighed, and separated into two groups of five. One group was used for protein content determination and the other for isoproturon residue determination. In addition, the soil from each container was kept for isoproturon residue analysis.

2. Materials and methods 2.3. Earthworm growth rate 2.1. Earthworms Earthworms (L. terrestris L.) were collected around the Faculty of Sciences in Reims, France, in an area

After 7, 15, 30, 45, and 60 days of exposure the weight of earthworms was compared to controls from untreated soil using relative growth rates, which were

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determined using the Eq. (1) of Martin (1986), where W0 is the weight at the beginning of incubation and Wt the weight after t days of exposure. Wt relative growth rate = ln × 100 (1) W0 2.4. Determination of total soluble protein content of earthworms Five earthworms (approximately 5 g) from each container were homogenized in three volumes (15 ml) of 20 mM Tris–HCl buffer (pH 8, ice-cold) containing 20% glycerol, 2 mM mercaptoethanol, 6 ␮M leupeptin, and 0.5 mM phenylmethylsulphonyl-fluoride (PMSF). The homogenate was then centrifuged at 18,000 × g for 40 min at 4 ◦ C. After the upper fatty layer was discarded the supernatant was taken. The protein concentration of the supernatant was determined by the dye binding method according to Bradford (1976). 2.5. Isoproturon extraction 2.5.1. Soil A 5 g sample of moist soil was taken from the sacrificed containers after different times of incubation. It was placed in a conical flask with glass stopper and isoproturon was extracted with 15 ml methanol using a mechanical shaker for 120 min. Extraction was repeated twice more with methanol. Pooled extracts were then centrifuged at 10,000 × g for 5 min at 4 ◦ C and transferred to a volumetric flask. The final volume was recorded and the methanolic extract was stored at 4 ◦ C. 2.5.2. Earthworms Five earthworms (approximately 5 g after gut clearance) from each container were homogenized in 25 ml of methanol using a glass homogenizer immersed in ice. The homogenate was shaken with methanol on a mechanical shaker for 120 min, filtered and then washed twice with 15 ml of methanol. The combined extracts were then centrifuged at 10,000 × g for 5 min at 4 ◦ C and quantitatively transferred to a volumetric flask. The final volume was recorded, and the methanolic extract stored at 4 ◦ C. A bioconcentration factor (or biota-to-soil accumulation factor) was calculated as the ratio of the mean

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isoproturon concentration in the animals divided by the mean soil concentration for each treatment. 2.6. Isoproturon quantification and extraction efficiency Soil and earthworm extracts (20 ␮l) were analyzed with high performance liquid chromatography (HPLC) using a reversed phase column (Kromasil, C18 100 Å, 5 ␮m, 250 mm × 3 mm, CIL-Cluzeau, Sainte Foy la Grande, France). Elution of isoproturon was performed isocratically with acetonitrile and water (2:1). Isoproturon was detected and quantified by monitoring the UV absorbance (200–300 nm) with a diode array detector (UVD 340, Gynkotek Dionex, Voisins le Bretonneux, France). Standard solutions of technical isoproturon (purity 99.5%, CIL-Cluzeau) were prepared and injected to obtain a standard curve of peak area versus isoproturon concentration (external standard method). The rate of recovery was estimated by the addition of 1 ml methanol containing pure isoproturon (1 ␮g/ml) to 5 g soil or 5 g earthworms. The extraction procedures were carried out as above. The average rate of isoproturon recovery, based on three replicates, was 89.5 ± 1.2%. The recovery rate was taken into account when calculating residue concentrations. 2.7. Data reliability Two independent experiments were conducted. Data presented are mean ± standard deviation (S.D.). Data were analyzed with one or two way analyses of variance and a pairwise multiple comparison procedure (Student–Newman–Keuls method) was used to compare treatments and duration of exposure. Sigma-Stat Software (Version 2.03, SPSS Inc., Chicago, USA) was used and statistical significance was set at P < 0.05. 3. Results and discussion 3.1. Residues of isoproturon in soil and earthworms Residues of isoproturon were detected in soil and earthworm tissue after 7, 15, 30, 45, and 60 days of exposure (Figs. 1 and 2).

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Fig. 1. Changes over time in isoproturon residue concentration in soils containing L. terrestris. Soils were treated with various concentrations of the herbicide. (A) Absolute values. Data for t = 0 day were deduced from applied concentrations. (B) Relative values. Bars represent standard deviations, n = 4.

In the soil, the concentration of isoproturon was not stable (Fig. 1A), it decreased significantly (P < 0.001) throughout the experiment. The rate of isoproturon decrease in soil corresponded more or less to first-order

kinetics and was rather slow. Indeed, the concentration decreased from 1.4 to 0.81±0.08 g/kg soil for the highest concentration and from 0.5 to 0.22±0.10 g/kg soil for the lowest concentration tested after 60 days.

Fig. 2. Changes over time in isoproturon concentration in earthworms (L. terrestris) placed in soils containing various concentrations of the herbicide (A) and bioconcentration factor of isoproturon in the worms (B). Bars represent standard deviations, n = 4.

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In the field the disappearance rate is higher; DT50 was reported to vary from 6.5 to 30 days (Walker et al., 2001). This may be due to the fact that leaching and runoff, which play an important part in the disappearance (Lecomte et al., 2001), were absent in our experiment. The fact that the soil had not received any pesticide treatment for over 35 years may also be involved, since it has been shown that soils with a phenylurea treatment history contain a microflora that can utilize isoproturon as nutrient source (Cullington and Walker, 1999). Furthermore, the concentrations used in our experiments mimic an accidental spill and were much higher than usual field concentrations. Indeed, assuming a herbicide distribution in the top 10 cm of the soil and a soil bulk density of 1.5 kg/l, the recommended field rate of 1.5 kg/ha is equivalent to an initial concentration of 1 mg/kg soil. Furthermore, statistical analysis of the relative isoproturon residues in the soil (percentage of applied, Fig. 1B) showed that the disappearance of isoproturon is significantly (P < 0.001) slowest for the highest initial concentration (1.4 g/kg soil), suggesting that the rate of isoproturon disappearance was faster with low concentrations than with higher concentrations (Fig. 1B). This was in agreement with the work of Helweg et al. (1998) in which biodegradation was very low at 5000 mg/kg soil while it was rapid at lower concentrations. Since degradation of isoproturon in soils has been reported to take place through a metabolic process (Cullington and Walker, 1999), it may be proposed that the highest concentrations used in our experiments may have been toxic to the soil microflora as has been suggested for mecoprop (Helweg et al., 1998). In earthworms, the presence of isoproturon was not simply due to contaminated soil in the animals since they were allowed to void gut content before extraction of the pesticide. Absorption of pesticides by earthworms may occur via ingestion but also by dermal uptake of lipophilic molecules because of the permanent contact between the worms and the soil interstitial water (van Straalen and van Gestel, 1993; Belfroid et al., 1995). The latter route is probably important in the case of isoproturon since its relatively low adsorption coefficient (KOC = 120 l/g; Lafrance et al., 2001) suggest that availability was not limited by adsorption of the pesticide to soil particles and its octanol/water partition coefficient (log KOW = 2.5) indicates a moderate hydrophobicity. As may have

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been predicted from the model of Widianarko and van Straalen (1996) describing internal body concentration of pesticide, isoproturon accumulation in earthworms displayed one “peak” (Fig. 2A). First, a rapid accumulation of the herbicide was observed in the worms to reach maximum concentrations of 0.27–0.52 ␮g isoproturon/g earthworm after 15 days of exposure for 0.5–1.4 g/kg soil, respectively. Thereafter, concentrations in the worms decreased slowly down to 0.07–0.31 ␮g isoproturon/g earthworm after 60 days of exposure and the difference between 7 and 60 days was no longer significant (P > 0.05). Such decrease may be attributed in part to the decrease of isoproturon availability in the soil and to the regular metabolism and elimination activities of the earthworms. These two parameters were described by two exponentials in Widianarko and van Straalen’s (1996) model. This fate of isoproturon in L. terrestris is also comparable to that of atrazine in another species of earthworm (Ismail et al., 1997a). For higher exposure concentrations (1.0–1.4 g/kg soil) the internal body concentration of the herbicide did not seem to follow the model exactly. Indeed, for these concentrations a lag phase was observed, the body concentration increase was slow during the first 7 days (Fig. 2A). Reduced food intake to avoid the pesticide may be hypothesized. Isoproturon availability in the soil can be invoked to explain in part the decrease of isoproturon concentration in the animals. When compared with other authors (e.g. Staak et al., 1998) the bioconcentration factor was very low in our study (Fig. 2B). Nevertheless, it appears that the bioconcentration factor increased rapidly after 15 days when the worms were exposed to low concentrations of the herbicide (0.5 and 0.8 g/kg soil) and it decreased thereafter (Fig. 2B). In contrast, for high soil concentrations of the herbicide the bioconcentration factor remained low and constant (between 0.3 and 0.5 mg/g). After 15 days of exposure the highest bioconcentration factor was then observed for the lowest concentration while it was low for the highest soil concentration. These results suggest that isoproturon accumulation in the worm is not a linear function of the soil concentration. Other mechanisms may be involved. Comparable results have been reported with various animals exposed to different pesticides (Streit, 1992; Streit, 1998). The decrease in the bioconcentration factor with increasing environ-

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Fig. 3. Effects of various concentrations of isoproturon on the relative growth rates of earthworms (L. terrestris) in soils treated with the herbicide. Bars represent standard deviations, n = 4.

mental pesticide concentration may be explained by a reduction of absorption. For example, it has been demonstrated that organisms can develop bioprotection mechanisms such as microvilli fusion or mucus secretion that limit the absorption of xenobiotics by the intestine (Streit, 1998). Bioconcentration may be impaired if the organism is exposed to toxic concentrations of xenobiotics (Cleveland and Hamilton, 1983; Streit, 1998). Toxicity may then explain why bioconcentration is low for high concentrations at all time and becomes low for low concentrations after a long exposure time (30 days): a less functional organism would reduce its feeding activity and thereby the absorption of isoproturon. This was confirmed by the reduction of growth rate observed (cf. see below). 3.2. Mortality No mortality was observed after 60 days of exposure to 0.5, 0.8, 1.0, 1.2, and 1.4 g of isoproturon/kg soil; thus the LC50 values after 60 days must be greater than 1.4 g/kg soil. We can conclude that isoproturon was non-toxic to mature earthworm. Another species of earthworm, Eisenia fetida, is also little affected by this herbicide (LC50 > 1 g/kg soil; Tomlin, 2000).

3.3. Effect of isoproturon on the growth rate of mature earthworms Under our experimental conditions, the growth rates of untreated worms were positive and increased significantly during the 60 days of the experiments (Fig. 3). The biomass of the worms increased from 9.22 ± 0.74 to 10.40 ± 0.16 g/10 worms after 60 days. In contrast, worms from isoproturon treated soils were significantly (P < 0.001) different from the controls. As indicated by the negative growth rates, they lost weight. The general tendency of the results showed that the decrease in weight was time and concentration dependent over the 60 days of the experiments. This was confirmed by the two way analysis of variance, which indicated significance for both parameters, and for their interaction (P < 0.001). Indeed, longer exposure time and/or higher concentration of isoproturon in the soil (especially between 0.5 and 1.0 g/kg soil) resulted in lower growth rate. After 60 days of exposure, the biomass of treated worms ranged between 9.57 ± 0.67 and 7.67 ± 0.34 g/10 worms, that is 10.1–27.9% less than the controls. Similarly, a herbicide formulation of amitrole and diuron (a phenylurea) was not lethal but reduced growth of L. terrestris under

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Fig. 4. Effects of various concentrations of isoproturon on the total soluble protein content of earthworms (L. terrestris) in soils treated with the herbicide. Bars represent standard deviations, n = 4.

some soil conditions (Bauer and Römke, 1997). Several other authors have shown that pesticide exposure may result in a decrease in earthworm growth rate: for example, 50 mg/kg of diflubenzuron in treated soil reduced the biomass of L. terrestris by 84% after 4 weeks of exposure (Ahmed et al., 1991). Also Ismail et al. (1997b) found a decrease in biomass for another earthworm (Aporrectodea caliginosa) exposed to pesticides (aldicarb, cypermethrin, atrazine, profenofos, metalaxyl and chlorfluazuron) after a period of 4 weeks. Reduction in growth rate was correlated with reduced feeding activity in an isopod exposed to endosulfan and it was suggested that reduced feeding may have been a strategy to avoid the pesticide (Ribeiro et al., 2001). Likewise, it may be proposed that the worms reduced their food intake to avoid contamination by isoproturon as confirmed by the stable bioconcentration factor (Fig. 2B). 3.4. Effect of isoproturon on total protein content of mature earthworms Throughout the 60 days of the experiment, control earthworms presented a stable protein content that ranged between 37±1.08 and 39.49±1.13 mg/g fresh

weight (Fig. 4). In all isoproturon treated worms a significant (P < 0.001) reduction in total protein content was observed. Although some concentration dependency may be observed, especially after 15, 30, and 45 days, no time dependency was found. There was a significant difference (P < 0.05) only between 7 and 15 days and between 7 and 30 days. This contrasted with the growth rate results and no direct correlation was found between the two parameters. Several authors have shown that reduction in worm protein content was one of the primary toxic effects of various pesticides. Ismail et al. (1997b) found that reduction in the total protein content of earthworm (A. caliginosa) might be the primary effect of chlorfluazuron, while it seems to be a secondary effect for other pesticides (cypermethrin, aldicarb, profenofos, atrazine and metalaxyl). This did not seem to be the case with isoproturon because reduction in protein content did not depend on time of exposure. Since it appears that the lower growth rate of the worms may be due to less food intake, the reduction in protein content may be ascribed to a catabolism of proteins in response to worm energy demand as suggested for an isopod in response to parathion (Ribeiro et al., 2001).

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Both growth rate and protein content of earthworms were proposed as biomarkers of exposure to pesticides (Ismail et al., 1997b; Mosleh et al., 2003). In the case of isoproturon it appeared that only growth rate may be used as a biomarker.

4. Conclusions At the concentrations tested, isoproturon degradation in soil was relatively slow since 50% of the initial concentration persisted after 60 days. This persistence of the pesticide in soil increases the risk of contamination of earthworms and other soil organisms after a herbicide spill. Accumulation of isoproturon in earthworms occurred during the first 15 days of exposure; the decrease in concentrations towards the end of the experiment may be related to the development of degradation processes in earthworms. Isoproturon may be considered non-toxic to L. terrestris since no mortality was observed after 60 days of exposure to 1.4 g isoproturon/kg soil. However, the sublethal toxic effect of this herbicide is clearly shown by significant reduction in growth rate. Furthermore, this study showed that within the concentration range that was used, isoproturon disappearance from soil was slower when the concentration increased, and isoproturon dissipation from the soil was much slower at high concentrations than at recommended field rates. An accidental spill leading to relatively high herbicide concentration in soils may therefore have localized consequences very much different from those observed in the case of diffuse pollution resulting from usual application rates.

Acknowledgements This work was financed in part by the French Ministère de la Recherche et des Technologies through the program “Unité de Recherches Vigne et Vin de Champagne (URVVC) UPRES-EA 2069”. Y.Y. Mosleh was awarded a fellowship from the Government of Egypt.

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