Alpha-Chloralose Bait Formulations and Their Laboratory and Field Efficacy in Common Vole (Microtus arvalis) Trials

Abstract

The common vole (Microtus arvalis) is a major rodent pest in European agroecosystems, causing periodic outbreaks that result in substantial crop losses and pose potential public health risks. Rodenticides remain the most widely used method for population control; however, current phosphide-based formulations present challenges related to environmental safety and non-target species exposure. This study evaluated the palatability and efficacy of novel alpha-chloralose bait variations for common voles. Laboratory trials were conducted in three phases: (i) screening of non-toxic cereal carriers to identify highly palatable formulations, (ii) comparison of alpha-chloralose from two manufacturers to select the optimal active ingredient, and (iii) enhancement of palatability and attractiveness through incorporation of several attractants. Choice and no-choice feeding tests revealed that alpha-chloralose efficacy is strongly influenced by bait formulation and pellet size, with small pellets (3 mm) ensuring that a single pellet provides a lethal dose for an individual vole. In laboratory conditions, the highest mortality rate, 50% (n = 12), was observed in the bait containing the milkvetch attractant. Subsequent small-scale field trials demonstrated that this bait achieved efficacy (85%) comparable to commercial zinc phosphide bait (90%). The study confirms that alpha-chloralose, when incorporated into optimized bait matrices, could be a viable rodenticide that combines rapid, humane action with a reduced risk of secondary poisoning, making it a promising tool for integrated pest management strategies.

1. Introduction

The common vole (Microtus arvalis Pallas, 1778) is widely recognized as the most significant rodent pest of arable crops in Europe [1,2,3]. Substantial agricultural losses are primarily associated with periodic population outbreaks, reaching densities of 1000–2000 voles per hectare, which occur at intervals of approximately 2–5 years [1,2]. During these peak years, vole abundance also poses increased risks to human health, as the species serves as a reservoir and vector for multiple zoonotic pathogens, e.g., Bartonella, Francisella, Leptospira, and Borrelia [4,5,6,7]. To mitigate both agricultural losses and public health risks, various strategies have been implemented to control vole populations [3,8,9]. The most widespread approach across EU countries is the application of rodenticides, most commonly based on the acute toxin zinc phosphide [10]. Baits are typically placed directly into burrow entrances to minimize exposure to non-target organisms [11]. In addition to rodenticides, which can effectively suppress vole populations in extensive agricultural fields, only certain agrotechnical practices, such as tillage, have been shown to be sufficiently effective on a large scale [12,13]. On the other hand, the often-proposed biological control using mammalian and avian predators has yielded only limited results [14]. Moreover, the simultaneous use of chemical and biological control methods is impossible, since the common vole is the main food of many species of owls [15,16], which can lead to negative consequences for their populations. Rodenticides used on agricultural land are classified under the regime of plant protection products (PPPs) in the EU, as established by Regulation (EC) No 1107/2009. In the Czech Republic, products containing two acute active substances are currently authorized: specifically, zinc phosphide (as a bait) and aluminum phosphide (as a soil burrow fumigant).

The utilization of rodenticidal baits has been identified as a large-scale, accessible and economically acceptable method for the effective control of vole populations on agricultural lands [8]. Nevertheless, it is important to note that non-target species may also be exposed to the baits, resulting in unwanted poisoning [17,18]. The implementation of appropriate bait application technology serves to mitigate this risk. In practice, however, a number of complications may arise. When treating large fields, workers may cause misapplication of bait, e.g., by overdosing or accumulating pellets at the entrance to the burrow, which makes the bait easily accessible to game or birds. Additionally, voles may remove pellets from burrow entrances themselves, particularly during routine cleaning when they dig up feces and soil clods [19]. Using small-sized bait pellets or an active substance that is effective even at very low bait volumes can help mitigate these risks.

However, given the economic and temporal constraints associated with the registration of new active substances, the introduction of entirely new rodenticides is unlikely. Therefore, attention has turned to substances already authorized for rodent control, such as alpha-chloralose [20]. In the EU, it is currently approved as an active substance for use in biocidal products of product type 14 (rodenticides) under Regulation (EU) No 528/2012. These products are authorized exclusively for the control of the house mouse (Mus musculus Linnaeus, 1758) and only for indoor use [20].

As an acute rodenticide, alpha-chloralose acts as a narcotic by depressing the rodent’s nervous system and metabolism, leading to lowered body temperature. In colder environments, this can result in hypothermia and death, a mechanism particularly effective in small rodents with a high body surface area-to-volume ratio. Furthermore, alpha-chloralose is most effective at temperatures below 16 °C [21].

The most prevalent rodenticides are baits based on pellets or grain, comprising non-toxic food ingredients and a toxic active ingredient. The food ingredients are key components, ensuring attractiveness and palatability in environments with a surplus of food sources, while also suppressing any undesirable taste effects of the active substance. Food preferences vary not only between rodent species, but also between populations of the same species, depending on locally available and familiar foods [22]. In addition to common components such as cereals and forage, other substances like flavors and attractants may be added to enhance bait attractiveness. The precise composition of each bait is unique, with recipes and preparation technologies closely guarded by each manufacturer.

Commonly, rodenticides for voles are formulated as pellets or poisoned cereal grains. However, baits in the form of poisoned cereal grains have also been found to be highly attractive to birds [18] and thus various types of granules or pellets, which are well accepted by voles, are now predominantly used [11]. In addition to the formulation of the bait, the presence of the active substance itself, its concentration, and the bait color have also been demonstrated to affect the palatability of the bait [11,23].

In view of the mandatory implementation of Integrated Pest Management (IPM) in the European Union, as required by Directive 2009/128/EC of the European Parliament and of the Council on the sustainable use of pesticides, and the current reliance on a limited number of highly toxic phosphide-based rodenticides for the control of the common vole, there is a clear need for alternative or complementary control strategies compatible with IPM principles. Within this framework, alpha-chloralose presents a strategic alternative to traditional acute toxins, as its narcotic mode of action potentially offers a more controlled impact on the environment and a reduced risk of secondary poisoning for non-target predators. Despite its recognized rodenticide properties, the application of this substance for common vole control remains largely unexamined. The novelty of this study lies in the systematic development of a bait formulation designed to overcome the innate taste aversion of common voles to this active ingredient. Our working hypothesis was that it would be possible to develop a new formulation containing alpha-chloralose with field efficacy comparable to conventional zinc phosphide-based rodenticides. Consequently, this study aimed to develop and evaluate a bait formulation based on alpha-chloralose as a potential alternative to metal phosphides. Specifically, the objectives of the laboratory trials were to (i) identify a suitable non-toxic food carrier, (ii) assess the applicability of alpha-chloralose as an active substance with appropriate toxicological and biological properties, and (iii) enhance bait attractiveness and palatability through the addition of attractants to overcome the aversive taste of alpha-chloralose. The efficacy of the developed bait was subsequently evaluated in pilot field trials and compared with conventional phosphide-based rodenticide baits.

2. Materials and Methods

The development of the new bait can be categorized into three successive phases. Initially, the selection of the non-toxic food components for the bait (formulation) was performed. In the subsequent phase of the study, active substances from different manufacturers were evaluated. Finally, additional attractant substances were added to mask the taste of alpha-chloralose and enhance the consumption of the bait to ensure the efficacy of the product. The efficacy and/or palatability of each bait was evaluated in common voles using standard laboratory feeding tests [24]. Initially, no-choice tests were performed to verify fundamental bait acceptance and confirm the effectiveness of the active substance concentration and the base carrier. Subsequently, choice tests were used to assess palatability and preference relative to a challenge diet, distinguishing between toxicological potency and behavioral acceptance [25]. Laboratory trials were conducted in three phases between 2020 and 2025 at the Animal Breeding Facility of Czech Agrifood Research Center. Further, the candidate baits were tested in small-scale field trials in 2025.

2.1. Experimental Animals

The common voles originated from the Animal Breeding Facility of Czech Agrifood Research Center. The animals were kept in standard plastic cages (30 × 15 × 15 cm) under standard laboratory conditions (temperature 19–22 °C, 55 ± 5% RH, 12:12 h light:dark cycle). The bedding material consisted of wood shavings, while hay was used for nest material and a plastic tunnel provided shelter. Water and food were provided ad libitum. Food was composed of the standard laboratory rodent diet (Biokron, Blucina, Czech Republic) and pellets for guinea pigs (De Heus, Bestovice, Czech Republic). The food was enriched with carrot, apple, fresh green leafy vegetables or herbs.

The tested animals were adult (3–5 months old), captive-bred voles of the first or second generation. We tested in total 71 males (mean body weight (±SE) = 28.73 ± 0.65 g; one male died from unknown reasons before the experiment) and 72 females (mean body weight = 23.93 ± 0.62 g). Voles were randomly assigned to test/control groups, n = 12 each (6 males and 6 females) [24].

2.2. Choice Tests with Non-Toxic Cereal Carriers

At the beginning of the experiment, voles were weighed and individually housed in the experimental wire-mesh cages (26 × 17 × 17 cm) for three consecutive days to allow acclimatization to the new housing conditions; laboratory diet and water were provided ad libitum. Subsequently, blank baits and laboratory diet (experimental) or only laboratory diet (control) were offered in two pots for a period of four days. Experimental blank baits were commercial pellets for common voles or synanthropic house mice and rats supplied by the manufacturers (PelGar Ltd., Prague, Czech Republic: blank bait 1 and 3; Agrochema druzstvo, Studenec, Czech Republic: blank bait 2); for more details see

Table 1. Characteristics of the proven blank commercial formulations in choice feeding tests on the common voles.

	Main Components of the Formulation	Wax Content (%)	Pellet Size (Diameter, mm)	Target Rodent Species
Blank bait 1	cereal meals (63%), sugar (6%)	20	2.5	common vole
Blank bait 2 *	cereal meals, dried alfalfa	0	4	common vole
Blank bait 3	cereals (67%), dried carrot (10%), sugar (5%)	10	2.5	house mouse, brown rat, black rat

* The percentage content of the main components cannot be disclosed, as it is proprietary to the manufacturer.

. The position of the two pots was changed daily to avoid any potential place preferences. Voles were checked daily, and remaining food was weighed (including spillage and crumbled food) and replenished. After the end of the experiment, all voles were returned to the breeding stock.

2.3. Verification of the Properties of the Active Substance

In the context of novel bait development, the cost of individual bait components represents a significant economic factor. The subsequent phase of the study involved a comparison of two available active substances of alpha-chloralose from two different manufacturers (Merck KGaA, Darmstadt, Germany and Look Pharmaceutical & Chemical Co., Ltd., Jinan, China; further referred to as DE and CHN, respectively), which differed in terms of economic cost. Due to the cost effectiveness of alpha-chloralose-CHN (approx. 10.5-times-lower costs), its other characteristics, including biological efficacy in in vivo tests and purity (declared ratio of alpha and beta isomers), were compared with alpha-chloralose-DE to assess its suitability for use in bait formulation.

Based on the results from the previous phase, blank bait 1 was selected as a palatable formulation for the active substances. The alpha-chloralose-DE and alpha-chloralose-CHN pellet baits (3 mm diameter pellets) were prepared in two concentrations (2 and 4%) and tested in the no-choice feeding trials. The tested baits were prepared and supplied by the manufacturer PelGar Ltd.

Alpha-chloralose baits were tested at 16 °C, their optimal efficacy threshold [21], in the experimental thermal chambers (12:12 h light:dark cycle). The animals were weighed and individually housed in plastic cages equipped with filter paper, hay and the plastic tube. After the three-day acclimatization, the test baits were offered for a 24 h period within the no-choice design. After the bait exposure, the remaining bait was weighed (including spillage and crumbled food) and replaced with the laboratory diet for a further four days. Any behavioral changes and the day of mortality were recorded; surviving voles were euthanized by cervical dislocation.

2.4. Alpha-Chloralose Purity

Alpha-chloralose exists in alpha and beta isomeric forms, but only the alpha isomer is pharmacologically and toxicologically active. The beta isomer, which is also present in technical products, is widely regarded as being biologically inactive [20]. The purity of both alpha-chloralose substances (DE, CHN) was verified using liquid chromatography with tandem mass spectrometry (LC/MS/MS) in a service chemical laboratory at the Faculty of Chemistry at Brno University of Technology.

Instruments: UHPLC Agilent 1290 (Santa Clara, CA, USA) and Bruker EVOQ (Billerica, MA, USA);

Column: Acquity UPLC BEH (2.1 × 50 mm; 1.7 μm);

Mobile phase: 0.5 mL/min, 0.1% HCOOH:ACN 90:10, gradient within 5 min 0.1% HCOOH:ACN 5:95 + 2 min post run;

LC/MS/MS: negative MRM mode, Q: 307- 188.9 (5eV), q: 307- 161.2 (5eV);

A total of 500 mg of alpha-chloralose sample was weighed. The sample was quantitatively transferred to a 100 mL volumetric flask and dissolved in approximately 70 mL of 50% methanol by shaking for 2 min and then sonicated for 10 min. After cooling to room temperature, the volumetric flask was made up to the mark with 50% methanol. The sample for analysis was filtered through a nylon syringe filter with a pore size of 0.22 μm, diluted 100,000 times and analyzed.

To ensure accurate determination of alpha isomers, a calibration curve was created with a range of 0.5–100 ng/mL of alpha-chloralose analytical standard in 50% methanol. Each sample was analyzed three times.

2.5. Choice Tests with Toxic Baits

As comparable values of purity and biological efficacy were achieved for the two active substances, alpha-chloralose-CHN was selected as the cheaper active ingredient for the novel bait formulation. Subsequent efforts concentrated on the incorporation of taste attractants into the fundamental recipe to enhance attractiveness and palatability of the bait. The bait mixture was enriched with additional ingredients and higher concentrations of alpha-chloralose were also tested to enhance the efficacy of the baits. The dosage of alpha-chloralose was recalculated according to the measured concentration of the alpha isomer to achieve the desired final concentration of the active alpha isomer in the bait [20]. Three baits (3 mm pellets) were offered with differing contents of alpha-chloralose, sugar, vanilla and milkvetch (for details see

Table 2. Mortality and bait consumption in choice tests with tested bait formulations. The number of voles analyzed for consumption was lower due to the contaminated bait (see the corresponding values in brackets).

Bait (Alpha-Chloralose Concentration)	Additive Ingredients	Mortality	Bait Consumption Mean ± SE (mg of Bait/g b.w.)
B1 (5%)	sugar 15%	3/12	11.3 ± 2.1 (6/12)
B2 (4%)	sugar 10%, vanilla 1%	4/12	13.4 ± 1.5 (8/12)
B3 (4%)	sugar 10%, milkvetch 2%	6/12	12.0 ± 2.2 (11/12)

The absence of superscript letters indicates no statistically significant differences (p > 0.05).

).

The efficacy of the baits was evaluated in a series of choice feeding trials. As the toxicity of alpha-chloralose is temperature-dependent, a low temperature of 6 °C was selected for the experimental trials. This value was selected as the lower temperature threshold for the autumn and winter months, when the application of alpha-chloralose could be typically performed. The experiments were conducted in thermal chambers, after a 14-day acclimatization period, which was extended due to low-temperature conditions. Following the acclimatization, the animals were individually housed in plastic cages equipped with filter paper, shredded paper and the plastic tube. The baits were offered for 2 days in the choice design with a challenge (attractive) diet of rolled oats [26]. After 24 h, the remaining bait was weighed and replaced with a fresh one and the mortality rate was recorded. The procedure was repeated on the following day, then the bait and the challenge diet were removed and replaced by a laboratory diet. The animals were observed for the next three days and then euthanized. Any behavioral changes and the day of mortality were recorded. Concurrently, six control pots containing an equivalent quantity of baits B1-B3 (3.1 ± 0.7 g) were placed in the thermal chambers to monitor the weight change (i.e., the water content) during the bait exposure.

When the experiments were being prepared, it became apparent that in many cases it was impossible to determine the consumption of rolled oats. This was due to the fact that the oats were crumbly, contaminated by urine and feces and had also absorbed moisture. In order to resolve this issue, two control groups of voles were established. The first group was fed a laboratory diet to demonstrate normal food intake (the consumption was assessed), while the second group was fed a diet of rolled oats to confirm acceptance of this challenge diet (food intake could not be determined); both diets were offered for 2 days.

2.6. Small-Scale Field Trials

The efficacy of the most successful baits (B2, B3), in comparison with a standard rodenticide used as a control, was further examined in pilot small-scale field experiments. The experimental area was located in a 20 m wide strip of alfalfa (Medicago sativa) near Jedomelice (Central Bohemia, Czech Republic). Two independent trials were conducted during two periods (October and December 2025) differing in vole activity.

In each trial, nine small plots (5 × 5 m) were established, monitored and treated. Vole population density was assessed using a pre-treatment census based on the number of burrow entrances reopened within 24 h. Briefly, all burrow entrances in each plot were initially counted and plugged, and the number of reopened entrances was recorded after 24 h.

Next, plots were randomly assigned to either the experimental or control group and treated with each bait in three replicates: experimental plots with alpha-chloralose bait B2 (vanilla-flavored) or B3 (milkvetch-flavored), and control plots with the standard commercial rodenticide Ratron^® GW (wheat bait containing zinc phosphide). A defined amount of alpha-chloralose-based bait (0.43 ± 0.01 g) was applied directly into reopened burrow entrances using a manual electric precision dispenser that enables accurate application of small rodenticide granules. The dispenser was developed as part of a research project that focused on the development of alpha-chloralose-based baits. Ratron^® GW bait (Frunol Delicia GmbH, Delitzsch, Germany) was applied in accordance with the product label (5 grains/hole) using a commercial dispenser (Ekolas SK Ltd., Paderovce, Slovakia). Mean temperatures during the 24 h period post-application were 11.4 °C (range 8.3–20.9 °C) and 2.3 °C (range 1.3–6.0 °C) for trials 1 and 2, respectively. After application, all plots and their surrounding areas were inspected twice daily for carcasses over the following two days. Treatment efficacy was evaluated seven days after application using a post-treatment census based on the number of reopened burrow entrances within 24 h, following the same procedure as the pre-treatment census.

2.7. Statistical Analysis

Individual bait/food consumption per day was expressed per gram of individual body weight (b.w.) at the start of the experiment. Additionally, in the choice tests with toxic baits, the mean weight change in the bait in the control pots was considered to calculate the bait consumption. Bait consumption was further evaluated by ANOVA and Tukey post hoc tests. Palatability was expressed as the proportion of bait consumed relative to total food intake.

In field trials, the efficacy of each treatment was assessed as percentage reduction from pre-treatment to post-treatment and further evaluated by ANOVA and Dunnet post hoc tests.

Statistical analyses were performed using Statistica software version 14.0.0.15 [27].

2.8. Ethical Note

All animal procedures were conducted in accordance with EU Directive 2010/63/EU for animal experiments, and ethical approval was obtained from the Institutional Animal Care and Use Committee of the Czech Agrifood Research Center (CARC). The experimental protocol was approved by the Ministry of the Environment of the Czech Republic (permit numbers MZP/2021/630/7, MZP/2023/630/2219 and MZP/2025/630/659).

3. Results

3.1. Choice Tests with Non-Toxic Cereal Carriers

Voles consumed different amounts of the blank baits (F_(2,29) = 7.76, p = 0.002). The factor sex and the sex*blank bait interaction did not affect consumption (F_(1,29) = 0.91, p > 0.05 and F_(2,29) = 0.48, p > 0.05, respectively). Subsequent post hoc analyses indicated that blank bait 2 was perceived as less palatable than the other two blank baits. The preference for blank bait 1 was statistically significant (p = 0.001), while only a similar trend was observed for blank bait 3 (p = 0.07). Voles in the control group ingested more of the lab diet than voles in all experimental groups (F_(3,39) = 5.43, p = 0.003). Similar results were obtained for palatability that differed among blank baits (F_(2,32) = 5.26, p = 0.01); post hoc tests showed significance between blank bait 1 and 3 (p = 0.01). For all means, see

Table 3. Mean (±SE) daily consumption of food in the choice feeding tests with blank baits.

		Mean Daily Consumption (mg of Bait/g b.w.)
Blank Bait	n	Blank Bait	Lab. Diet	Palatability (%)
Blank bait 1	11	153.6 ± 13.8 a	39.3 ± 3.6 a	78.5 ± 2.5 a
Blank bait 2	12	87.5 ± 11.6 b	67.1 ± 9.0 a	55.7 ± 5.9 b
Blank bait 3	12	126.1 ± 9.4 ab	56.7 ± 17.3 a	71.8 ± 5.8 ab
Control	12	-	294.8 ± 11.6 b	-

Different letters indicate statistically significant differences according to ANOVA followed by Tukey’s post hoc test, p < 0.05.

.

Based on the above results, blank bait 1 was selected as a palatable formulation for the subsequent phase of bait development.

3.2. Verification of the Properties of the Active Substance

The bait consumption of five voles could not be determined due to the bait being contaminated with urine. Consequently, only the consumption of 43 voles was subjected to further analysis. The consumption of bait differed significantly among bait types (F_(3,35) = 16.62, p < 0.001), whereas no significant differences were found between sexes (F_(1,35) = 3.76, p = 0.06). The sex*bait interaction did not affect bait consumption (F_(3,35) = 1.27, p > 0.05). The results of post hoc tests are shown in

Table 4. Mortality and bait consumption in no-choice tests with alpha-chloralose of different origins. The number of voles analyzed for consumption was lower due to the contaminated bait (see the corresponding values in brackets).

Alpha-Chloralose Origin and % in Bait	Test Temperature (°C)	Mortality	Bait Consumption Mean ± SE (mg of Bait/g b.w.)
CHN (2%)	16	3/12	75.7 ± 9.5 c (10/12)
DE (2%)	16	4/12	49.5 ± 9.3 a (10/12)
CHN (4%)	16	6/12	25.7 ± 3.1 ab (11/12)
DE (4%)	16	4/12	15.9 ± 4.0 b (12/12)

Different letters indicate statistically significant differences according to ANOVA followed by Tukey’s post-hoc test, p < 0.05.

.

On the other hand, mortality was determined in all 48 voles. Most mortality occurred on the first day of the experiment (n = 17), with only four voles dying on the second day of bait introduction. The highest mortality was achieved in the 4% alpha-chloralose-CHN bait (Table 4). Despite the increased consumption of the 2% bait, this did not result in higher mortality (see Table 4).

LC-MS/MS analysis revealed the purity (proportion of the alpha isomer) of both alpha-chloralose-CHN and alpha-chloralose-DE, which were found to be 83% and 88%, respectively. These values are both in accordance with the value of 80%, a minimal threshold that has been established by the producers.

Since both biological trials with voles and purity analyses revealed comparable results for both active substances, the more affordable (10.5-times-lower costs) alpha-chloralose-CHN (4%) was selected for the preparation of additional formulations.

3.3. Choice Tests with Toxic Baits

The analysis of bait consumption was performed with the reduced number of experimental voles (n = 25). In the reduced dataset, the effect of the tested bait on consumption was only evaluated (the factor sex could not be evaluated) and no significant variation among the baits was detected (F_(2,22) = 0.41, p > 0.05).

In choice trials, the mortality rate was determined for all voles that were included in the study (n = 36). The lowest mortality was detected for the B1 formula with a higher (5%) concentration of alpha-chloralose. The B2 and B3 formulae with 4% alpha-chloralose reached 33 and 50% mortality (see Table 2). Most mortality occurred on the first day of the experiment (n = 12), with only two voles dying on the second day of bait introduction. Voles consumed similar amounts of the tested baits; bait intake was assessed in 25 voles (Table 2). Control voles (the first control group) consumed 294.8 ± 11.6 mg/g of laboratory diet daily. The second control group, fed rolled oats, accepted this diet well and survived the two-day feeding period but their food intake could not be exactly determined.

3.4. Small-Scale Field Trials

In trial 1, total pre-treatment vole activity was 40.7 ± 4.9 holes per plot. Mean efficacy significantly differed among treatments (F_(2,6) = 8.05, p < 0.05). Dunnett’s post hoc test, with Ratron^® GW as the reference group, revealed that while the efficacy of B3 (52.7%) was comparable to Ratron GW (P > 0.05), the B2 bait showed an increase in vole activity (−7%); for more results see

Table 5. Number of common vole holes (mean ± SE) reopened during field trials and efficacy of tested rodenticide baits (n = 3 plots per treatment).

Bait	Trial 1			Trial 2
	Pre-Treatment	Post-Treatment	Efficacy	Pre-Treatment	Post-Treatment	Efficacy
B2 (vanilla)	29.0 ± 8.7	30.0 ± 7.5	−7.0% *	13.7 ± 2.2	4.3 ± 2.8	64.3%
B3 (milkvetch)	52.3 ± 7.8	38.0 ± 4.9	25.7%	12.0 ± 1.0	1.7 ± 0.9	84.9%
Ratron® GW	40.7 ± 3.5	21.7 ± 5.0	47.9%	14.7 ± 4.3	1.3 ± 0.7	89.7%

* Indicates statistically significant differences compared to the reference group (Ratron^® GW) according to ANOVA followed by Dunnett’s post hoc test, p < 0.05.

. One carcass was found during post-treatment inspection of the plots.

The second trial started with a lower baseline activity (mean of 13.4 ± 1.5 holes per plot). Mean efficacy reached 64.3%, 84.9% and 89.7% for B2, B3 and Ratron^® GW baits, respectively (Table 5). No significant differences in efficacy were found among the treatments (F_(2,6) = 0.8, p > 0.05), suggesting that both experimental baits performed comparably to the commercial standard Ratron^® GW. No carcasses were found during this trial.

4. Discussion

This work represents the first systematic attempt to develop an alpha-chloralose-based bait specifically targeting the common vole. Prior to our study, it was well-established that alpha-chloralose serves as a distinct tool within the biocide and rodenticide toolkit across both EU and non-EU regions. A compelling reason for our selection of this active ingredient is its toxicological profile. From this perspective, the compound is valued for its unique mode of action—the rapid induction of narcosis followed by fatal hypothermia. This mechanism is frequently cited in the literature as a more humane alternative to the prolonged distress associated with anticoagulants [28]. However, despite the rapid onset of clinical signs (5–20 min), a point of contention remains regarding whether this “humane” profile is effectively balanced by practical efficacy [20,29].

The development of the bait followed a rigorous, stepwise, and sequential approach designed to address the inherent challenges of alpha-chloralose and bridge the gap between laboratory efficacy and field performance. This process began with choice tests using various non-toxic cereal carriers to identify a substrate with the high inherent appeal necessary to ensure that voles initiate feeding without hesitation. We then conducted a comparative analysis of alpha-chloralose from different global manufacturers to balance chemical purity and biological efficacy with the economic feasibility of large-scale production, followed by toxic choice tests to refine the bait’s composition. A primary focus during this stage was the integration of palatability enhancers and attractants to mask the active ingredient and prevent the early cessation of feeding. The final stage involved field-testing our prototype baits under realistic environmental conditions, where we benchmarked our formulation’s performance against the existing industry standard, specifically traditional baits based on zinc phosphide. The results achieved and the critical challenges identified during this experimental process are discussed within the context of specific topics in the following sections of this paper.

Choice tests with non-toxic cereal and saccharide carriers. Selecting a rodenticide matrix with high baseline palatability represents a key prerequisite for achieving efficacy across a broad spectrum of pest rodent species [30]. Both synanthropic rodents and other rodent taxa often exhibit similar taste preference patterns and may actively seek comparable bait types and food substrates [31]. In the present study, initial screening of non-toxic food carriers demonstrated high acceptance by common voles for two pellet types originally formulated for voles (78.5%) and for synanthropic rodents (71.8%). This high palatability is likely attributable to their composition based on cereal meals and sugars, which act as strong phagostimulants by signaling high caloric value [32,33]. Consequently, energy-dense substrates such as saccharides and processed cereals represent highly effective carriers in rodenticide formulations, as they can successfully compete with natural forage, particularly when supplemented with saccharides or sweeteners (sucralose) that help to overcome species-specific neophobia [34,35]. Based on the palatability results, blank bait 1 was selected as the reference palatable formulation for the subsequent phase of bait development. This choice enabled the experimental focus to shift toward testing different concentrations of alpha-chloralose while minimizing variability associated with bait acceptance [29].

Verification of the properties of the active substance. During the evaluation of alpha-chloralose formulations from two manufacturers (CHN, DE), concentrations of 2% and 4% were tested. Although baits containing 2% active substance were consumed in greater quantities, this increased intake did not result in higher mortality as the rapid mode of action requires a lethal dose to be consumed within an extremely short time window, typically before the onset of intoxication or narcosis [21,36]. Consequently, the 4% formulation was selected for further bait optimization, in line with previously reported effective concentrations for house mice [20].

Choice tests with toxic baits and palatability enhancement. Determining and improving palatability remains particularly challenging for acute rodenticides, as sufficient bait intake must occur before the onset of intoxication. In our experiments, this limitation was reflected by the consistently low quantities of bait ingested by voles, a feeding pattern that mirrors observations in other species such as the house mouse [29,37]. In these species, rapid intoxication—especially under field conditions where a great variety of alternative food is available—may affect selection and/or total intake [29]. Therefore, our choice test results further support the hypothesis that alpha-chloralose possesses a strong, aversive taste profile, as the lowest mortality (25%) was recorded for the B1 formulation containing the highest (5%) concentration of the active ingredient. Our findings confirm that a 4% concentration represents the optimal threshold for Microtus arvalis providing a critical balance between sufficient toxicity and acceptable palatability; this allows for higher ingestion rates compared to the 5% variant while maintaining greater lethal potential than the 2% formulation (tested in the no-choice trials).

Given that the basic cereal formulations achieved limited mortality, subsequent efforts focused on improving bait palatability through the incorporation of attractants. Previous research has indicated that specific botanical compounds can be highly attractive to voles [26]. Therefore, in this study, milkvetch (Astragalus spp.) was selected as a primary attractant due to its unique and rich composition of botanical chemical compounds and saccharides [38]. Although this enhancement did not lead to 100% mortality—reaching a maximum of 50% in choice tests—it represented the most successful formulation among all tested variants.

A primary challenge in our laboratory trials was the high rate of bait contamination by urine and feces, which led to the exclusion of several data points to maintain the accuracy of consumption measurements. This reduction in sample size, while necessary for data integrity, represents a limitation of the study by reducing the overall statistical power and the strength of the interpretation of consumption patterns at low temperatures.

Importance of bait formulation and pellet design. Bait formulation is a key factor influencing the acceptance and efficacy of rodenticides [30,39], and this is particularly critical for acute compounds with a rapid onset of action such as alpha-chloralose. In the present study, special attention was paid to the physical characteristics of the bait, with an emphasis on the development of small-sized pellets suitable for direct application into vole burrows. Pellets with a diameter of 3 mm were found to be sufficient, as voles consume only very limited amounts of bait. In other rodent species, particularly house mice, poor palatability of alpha-chloralose baits can often be overcome by soft bait paste formulations [40]. Soft baits are generally regarded as highly attractive and effective for a wide range of rodent species [41], including house mice [37]. However, such formulations are unsuitable for field situations that require direct placement of bait into small burrow systems, such as those of common voles, while also ensuring environmental stability and eventual degradation of the bait material.

Small-scale field trials. Small-scale field trials demonstrated that the efficacy of experimental alpha-chloralose baits was strongly influenced by initial vole activity (number of opened holes). Under conditions of high activity (40.7 holes per plot), marked differences among formulations were observed. Bait B2 (vanilla-flavored) failed to provide control, which may indicate (under the tested field conditions) low bait attractiveness, insufficient intake of the active substance, or increased bait avoidance under high population pressure. In contrast, the newly proposed bait B3 (milkvetch-flavored) achieved efficacy comparable to the reference product Ratron^® GW (based on zinc phosphide), suggesting that an appropriate alpha-chloralose formulation can provide effective control even with high vole numbers. With lower burrow activity (13.4 holes per plot), differences among treatments disappeared and all tested products showed high efficacy. This supports the assumption that alpha-chloralose could perform particularly well under lower vole activity, where bait acceptance is higher and competition for food resources is reduced. The lack of significant differences (in small-scale field conditions) between experimental baits and the commercial reference under these conditions indicates that optimized experimental formulations may represent viable alternatives to registered products.

The field trials were conducted on small-scale plots (5 × 5 m), a design necessitated by several critical constraints encountered during the final season of the project. The availability of suitable sites was limited to specific vole-infested areas where both farmers and state authorities granted permission for the simultaneous application of registered zinc phosphide baits and experimental, currently unregistered alpha-chloralose formulations. Furthermore, the experimental site was restricted to an alfalfa strip approximately 20 m wide. To maintain statistical power under these conditions, we prioritized plot homogeneity regarding vole activity and ensured sufficient replication within a single season. This approach was essential for the evaluation of bait performance under standardized field conditions. While we acknowledge that the absence of the perimeter buffer zones recommended by EPPO guidelines [42] may allow for migration from surrounding untreated areas, the comparative validity of the results remains. Since all treatment plots were subjected to identical environmental conditions and migration pressure from the surrounding area, the relative performance and efficacy rankings of the tested baits are robust. Although small-scale plots may typically, to some extent, lead to an underestimation of absolute mortality rates due to reinvasions (as was probably the case with the B2 bait in trial 1, where no reduction in vole activity was found), the fact is that our optimized experimental formulations achieved efficacy levels comparable to the registered commercial reference product. While larger experimental areas with treated buffers would be ideal for definitive registration trials to minimize edge effects, the chosen methodology provided the controlled environment necessary to demonstrate that these optimized formulations represent a viable alternative to existing products under identical conditions.

IPM considerations and future research directions. Overall, the present results demonstrate that the identification of an optimal bait formulation for common voles is a complex and multifactorial task influenced by a combination of biological, behavioral and technological factors. In addition to bait composition, application technology plays a crucial role in determining field efficacy, as highlighted by Jacoblinnert et al. [43], who demonstrated significant differences among baiting strategies in vole management. From an environmental risk perspective, alpha-chloralose offers several important advantages. Due to its acute mode of action and the very small quantities of bait ingested by voles, the risk of secondary poisoning of predators and scavengers through consumption of intoxicated rodents is substantially reduced. However, the potential hazard to non-target bird species remains a concern and has been documented previously [21], underlining the need for carefully controlled application methods and further refinement of bait formulations. Under laboratory conditions, mortality of 50% was achieved in selected formulations, and in small-scale field trials several efficacy values were comparable to those obtained with currently used products based on toxic phosphides. While these results are encouraging, they also confirm that further optimization is necessary before broader application can be recommended.

There are several potential pathways for optimization. Although we did not conduct such experiments within the scope of this study, the existing literature allows us to hypothesize that one promising area for future research is the microencapsulation of alpha-chloralose and the further optimization of its distribution within the bait matrix [44,45]. Such techniques could effectively mask the taste or physiological onset of the active ingredient, potentially increasing the amount of bait consumed before the onset of narcosis and thereby improving overall field efficacy.

Alpha-chloralose’s environmental impact and pest welfare: IPM principles require that any negative environmental impact of rodenticide application be minimized. Although alpha-chloralose is inherently less toxic than zinc phosphide and lacks the cumulative potential associated with anticoagulant rodenticides, its environmental profile warrants careful consideration. During our field observations, no immediate adverse effects on non-target species were recorded among the collected carcasses. However, the comprehensive impact on non-target wildlife requires careful consideration, particularly with respect to avian predators such as owls, for which common voles may represent an important prey resource [15,16]. Alpha-chloralose has also been used as an avicide for pest bird control in some non-EU regions. This is particularly relevant because birds are generally more sensitive to alpha-chloralose than mammals; concentrations suitable for bird control have been reported to be around 2% [46]. Therefore, although alpha-chloralose may offer some advantages over anticoagulant rodenticides due to its acute mode of action and lower potential for bioaccumulation, its possible effects on birds cannot be overlooked. It is necessary to acknowledge that environmental risks exist and must be thoroughly addressed before the widespread introduction of this bait in open-field conditions.

Due to the acute mode of action of alpha-chloralose, target species typically consume only a small amount of bait before the onset of sedation. This restricted intake may result in lower residue levels in prey and could therefore reduce, but not eliminate, the risk to predators and scavengers. Documented cases of alpha-chloralose poisoning in domestic animals, particularly cats and dogs, show that non-target exposure remains relevant [47,48]. Moreover, secondary exposure has been demonstrated in domestic cats after ingestion of alpha-chloralose-poisoned mice [49], indicating that residues in intoxicated or dead rodents may be sufficient to affect mammalian predators or scavengers. The risk profile of alpha-chloralose therefore differs from that of anticoagulant rodenticides, which are persistent, bioaccumulative, and widely associated with secondary poisoning of predatory wildlife [49]. It also differs from zinc phosphide, which is rapidly converted to phosphine after ingestion and is generally considered to pose a relatively low residue-mediated secondary poisoning risk, although direct poisoning of non-target species through bait consumption remains an important concern.

Modern IPM strategies must increasingly address the complex relationship between the efficacy of pest control products and the welfare of the target species. Ethical considerations in vertebrate pest control emphasize the reduction in pain and distress, making the selection of rodenticides with a humane mode of action a priority. Due to its specific mechanism of action—inducing rapid narcosis followed by fatal hypothermia—alpha-chloralose is recognized as one of the few rodenticides that can eliminate harmful rodents both rapidly and humanely [28]. The onset of symptoms is notably fast, which contrasts favorably with second-generation anticoagulants, where the time to death is significantly longer and often accompanied by prolonged internal hemorrhaging. Furthermore, the clinical profile of alpha-chloralose provides an additional margin of safety regarding sublethal exposure. In cases where a lower dose is ingested, or environmental conditions prevent the onset of fatal hypothermia, the animal does not suffer permanent physiological damage; instead, recovery typically occurs after several hours as the substance is metabolized [20,29]. This characteristic underscores the potential of alpha-chloralose as a highly effective yet ethically acceptable tool within the framework of professional pest management, provided that bait formulations are optimized to ensure a lethal dose is consumed during the initial feeding bout.

5. Conclusions

In conclusion, this study provides a comprehensive experimental evaluation of alpha-chloralose-based baits, demonstrating both their potential and their current limitations in the control of the common vole (Microtus arvalis). Our findings indicate that alpha-chloralose should not be viewed as a simple and straightforward replacement for existing methods, but rather as a useful component within the framework of IPM. Recognizing that IPM strategies often utilize interventions that do not individually achieve 100% efficacy, this study demonstrated that—under specific experimental conditions—certain alpha-chloralose formulations could achieve efficacy levels comparable to traditional acute rodenticides based on zinc phosphide.

However, the moderate efficacy and variability observed during these trials suggest that these results should be interpreted with caution regarding their immediate readiness for broad-scale field application. The specific limitations of this study—specifically the small scale of the experimental plots and the resulting potential for migration—highlight the necessity for broader field validation. Our results clearly demonstrate that efficacy is influenced by bait formulation and local vole activity (e.g., 84.9% efficacy for the B2 formulation under conditions of lower baseline activity). These variables must be more precisely controlled and understood before large-scale implementation can be recommended.

Future research should prioritize delivery technologies, such as microencapsulation (similar to methods previously evaluated for synanthropic rodents), to mask the taste of the active ingredient and refine bait matrices to increase palatability. Such advancements would help ensure that a lethal dose is consumed during a single feeding bout. At this stage of research, alpha-chloralose baits should be viewed not as a breakthrough standalone solution, but rather as a complementary tool. Given that IPM relies on the synergistic effects of multiple, environmentally responsible measures, effective vole management is best achieved through a coherent system of varied interventions. While further optimization is required to enhance reliability, alpha-chloralose offers one of the few chemical alternatives for vole control that aligns with at least some aspects of modern standards for vertebrate pest management.