The Insecticide Imidacloprid Promotes Algal Growth in Absence of Zooplankton

Abstract

Imidacloprid, a systemic neonicotinoid insecticide, exerts its neurotoxic effects by binding to nicotinic acetylcholine receptors in the central nervous system. In this study, we examined the effects of commercial imidacloprid formulations on the growth of Chlorella vulgaris and other algal species, comparing these responses with those induced by plant hormones. Our results demonstrate that formulated imidacloprid stimulates C. vulgaris growth at concentrations as low as 7.82 μM, with a more pronounced effect than certain phytohormones. We observed similar growth-enhancing effects in other algal species exposed to imidacloprid. Notably, pure imidacloprid induced equivalent growth responses in C. vulgaris, confirming that the observed stimulation results from the active ingredient itself rather than formulation adjuvants. Given its insecticidal mode of action, potential worst-case aquatic contamination scenarios with imidacloprid may lead to significant increases in algal biomass through both direct (growth stimulation) and indirect (reduction of zooplankton grazing pressure) mechanisms.

1. Introduction

Imidacloprid, a systemic neonicotinoid insecticide, exerts its neurotoxic effects by binding to nicotinic acetylcholine receptors in the central nervous system of insects [1]. Beyond its insecticidal properties, growing evidence suggests that neonicotinoids may directly influence plant and algal physiology. While previous studies have documented increased crop yields following neonicotinoid application—even in the absence of insect pressure [2,3]—the underlying physiological mechanisms remain poorly characterized. Gonias et al. (2006) [3] proposed a reduction in stress temperature on cotton as a potential mechanism during imidacloprid treatments, although subsequent advancements in this area have been limited. More recently, Ford et al. (2010) [4] demonstrated that imidacloprid triggers salicylate-mediated defense responses in Arabidopsis thaliana. This finding is particularly intriguing given that salicylic acid, while generally inhibitory to vascular plant growth, has been shown to stimulate proliferation in algal species [5] and is now recognized as a novel phytohormone for microalgae like Chlorella sp. [6]. Further complexity arises from work by Lukaszewicz et al. (2019) [7], who reported that imidacloprid alters mitotic kinetics in the macrophyte Bidens laevis L., inducing significant increases in prophase cells, while reducing metaphase frequency.

The interaction between imidacloprid and algal systems remains underexplored, with most existing research focusing on microalgae for bioremediation purposes [8]. However, several studies have noted not just remarkable algal tolerance to imidacloprid, but actual growth stimulation at certain concentrations [9]. Freshwater ecosystems have been contaminated with imidacloprid at concentrations as high as 320 µg/L [10]. During routine ecotoxicological testing with Chlorella vulgaris, we made a counterintuitive observation: Significant growth enhancement occurred at high concentrations of commercial imidacloprid formulations. This prompted us to systematically test three hypotheses: (1) Imidacloprid induces growth stimulation comparable to known plant hormones in algae; (2) this growth-promoting effect is conserved across multiple algal species; and (3) the observed phenomenon is mediated by the active ingredient itself rather than formulation additives. Our findings may have important ecological implications, as imidacloprid contamination could potentially disrupt aquatic primary producer dynamics through direct growth modulation.

2. Materials and Methods

Assays were carried out in 10 mL glass tubes using Bold medium. The culture chamber was maintained at 24 ± 1 °C and 12:12-h photoperiod; pH media was always ≈7. Imidacloprid was obtained in two distinct commercial formulations (Punto 3.5 of Gleba^® and GlacoXan^®). Gibberellic acid, thidiazuron, and indoleacetic acid were procured from China as active principles (Figure 1). Statistical analyses were performed in R using linear mixed models with the packages nlme and emmeans. In general, as repeated measures were used in time, the models used in each assay followed this structure.

Model <- lme (Abs ~ Treatment x Day, random = ~1 | Tube)

Given that the variance was not equal across the different treatments, various models were constructed to account for this. The model with the lowest AIC and BIC values was selected; in all cases, the model with varIdent was finally chosen and used.

2.1. Assay 1: C. vulgaris Growth Under Different Imidacloprid Concentrations

Fifteen tubes with 8 mL of 1:10 Bold’s Basal Medium with C. vulgaris were utilized for treatments using the Gleba commercial formulation of imidacloprid Punto 3.5 (L.28161) purchased in Argentina. Treatments included control (0 mg/L) and 78.2 nM (0.02 mg/L), 0.782 μM (0.2 mg/L), 7.82 μM (2 mg/L), and 78.2 μM (20 mg/L) of imidacloprid. Treatments were conducted by triplicate and measured in spectrophotometer HACH DR5000 UV-Vis (Hach Company, Loveland, CO, USA) at 675 nm on days 0, 3, 4, 5, 7, and 10 post-treatments.

2.2. Assay 2: C. vulgaris Growth Under Imidacloprid and Plant Hormones Treatments

Eighteen tubes containing 8 mL of 1:10 Bold’s Basal Medium with C. vulgaris were employed for various treatments, including Control, Imidacloprid Gleba 78.2 μM, Imidacloprid GlacoXan (L.5074A purchased in Argentina) 78.2 μM, Thidiazuron 78.2 μM, Indolacetic acid 78.2 μM, and Gibberellic acid 78.2 μM. Triplicate treatments were evaluated at 675 nm on days 0, 1, 2, 3, 6, 7, 10, 13, 17, and 21 post-treatments. Additionally, one sample from each treatment was observed under 640× magnification using an inverted microscope after 24 h of sedimentation.

2.3. Assay 3: Growth Curves of Haematococcus pluvialis, Tetrademus sp., and Monoraphidium obtusa Under 7.82 μM of Imidacloprid Gleba Treatment

Isolated algae from Salta, Argentina, were cultivated in 8 mL of 1:10 Bold’s Basal Medium tubes. Treatments consisted of control and 78.2 μM of imidacloprid Gleba. Concentration was selected based on the highest observed growth effects on C. vulgaris, without signs of inhibition. Four replicates were performed for each treatment, and measurements were taken at 675 nm on days 0, 4, 6, 7, and 10.

2.4. Assay 4: Growth Response of C. vulgaris to Analytical Grade Imidacloprid

Due to the substantial cost difference between analytical-grade standards and commercial formulations, combined with funding limitations for scientific research in Argentina, we conducted a single confirmatory assay to establish whether the growth stimulation resulted from imidacloprid itself rather than formulation additives. To enhance treatment sensitivity, we used undiluted Bold’s Basal Medium (unlike previous experiments with 1:10 diluted medium). The assay employed imidacloprid analytical standard (Sigma-Aldrich, CAS 138261-41-3, PESTANAL^®, St. Louis, MO, USA) at 78.2 μM, with seven biological replicates each for the treatment and control groups over a 10-day exposure period.

3. Results

3.1. C. vulgaris Growth Under Different Imidacloprid Concentrations (Assay 1)

Figure 2 displays the growth curves of C. vulgaris exposed to increasing concentrations of imidacloprid (Gleba^® formulation) over a 10-day period. A linear mixed-effects model showed that treatment (F_4,10 = 83.56, p < 0.0001), time (F_5,50 = 137.91, p < 0.0001) and their interaction (F_20,50 = 46.19, p < 0.0001) significantly influenced C. vulgaris growth (

Table 1. Summary of the lineal mixed model obtained for the absorbance (675 nm) in C. vulgaris growth curves under different concentrations of imidacloprid Gleba^®. Statistically significant p-values (<0.05) are shown in bold.

	numDF	denDF	F-Value	p-Value
(Intercept)	1	50	1882.23	<0.0001
Treatment	4	10	83.56	<0.0001
Day	5	50	137.91	<0.0001
Treatment–Day	20	50	46.19	<0.0001

), confirming differential growth responses depending on both concentration and exposure duration. Tukey post hoc contrasts revealed no significant differences in absorbance between treated and control groups during the early stages (days 0–3). However, the highest concentration (78.2 μM) significantly enhanced algal growth starting from day 4 (p = 0.0241), with increasingly stronger effects observed on days 5 (p = 0.0016), 7 (p < 0.0001), and 10 (p < 0.0001) (

Table 2. Tukey contrasts between different concentrations of imidacloprid and the control group. Statistically significant p-values (<0.05) are shown in bold.

Day	78.2 nM	0.782 µM	7.82 μM	78.2 μM
0	0.9960	1.0000	0.9955	1.0000
3	0.9960	0.9363	0.8387	0.9987
4	0.8595	0.7276	0.0180	0.0241
5	0.9493	0.9995	0.0115	0.0016
7	0.9681	0.9871	0.0026	<0.0001
10	0.8595	0.9790	0.0005	<0.0001

). Similarly, the 7.82 μM treatment also promoted growth from day 4 (p = 0.0180) onward. By day 10, the absorbance in the 78.2 μM treatment group was on average 5.84 times higher than in the control group, indicating a marked and concentration-dependent stimulatory effect of imidacloprid on C. vulgaris growth.

3.2. C. vulgaris Growth Under Imidacloprid Formulations and Plant Hormones Treatments (Assay 2)

To compare the growth-promoting effects of imidacloprid and plant hormones on C. vulgaris, we conducted a 21-day assay using a standardized concentration of 78.2 μM for all treatments. The tested compounds included two commercial formulations of imidacloprid (Gleba^® and Glacoxan^®) and three plant hormones—gibberellic acid (GA), thidiazuron (TDZ), and indoleacetic acid (IAA)—to represent distinct hormonal modes of action. As shown in Figure 3 and Figure 4, the growth curves revealed differential responses among treatments. The linear mixed-effects model identified significant effects of treatment (F_5,17 = 369.92, p < 0.0001), time (F_10,170 = 176.09, p < 0.0001), and their interaction (F_50,170 = 144.20, p < 0.0001), indicating treatment-specific dynamics over time (

Table 3. Summary of the lineal mixed model obtained for the absorbance (675 nm) in C. vulgaris growth curves under 78.2 μM concentrations of imidacloprid in two commercial formulations, gibberellic acid, thidiazuron, and indolacetic acid. Statistically significant p-values (<0.05) are shown in bold.

	numDF	denDF	F-Value	p-Value
(Intercept)	1	170	3489.65	<0.0001
Treatment	5	17	369.92	<0.0001
Day	10	170	176.09	<0.0001
Treatment–Day	50	170	144.20	<0.0001

). Among the tested substances, C. vulgaris responded significantly only to gibberellic acid and the two imidacloprid formulations. GA induced an early growth promotion from day 3 (p = 0.0051), whereas Gleba^® and Glacoxan^® triggered significant responses later, from day 6 (p = 0.0004) and day 7 (p = 0.0015), respectively (

Table 4. Tukey contrasts between treatments and the control group. Statistically significant p-values (<0.05) are shown in bold.

Day	Indolacetic Acid	Thidiazuron	Gibberellic Acid	Imidacloprid Gleba®	Imidacloprid Glacoxan®
0	0.9990	0.9734	0.9989	1.0000	1.0000
1	0.9987	0.8953	0.9898	1.0000	0.9996
2	0.9960	0.9151	0.3336	0.9985	0.9928
3	0.9940	0.9536	0.0051	0.9907	0.9790
6	0.9944	0.8985	0.0026	0.0004	0.0551
7	0.9855	0.8181	0.0031	<0.0001	0.0015
10	0.9811	0.8181	0.0022	0.0001	<0.0001
13	0.9734	0.7894	0.0020	<0.0001	<0.0001
15	0.9108	0.7639	0.0022	<0.0001	<0.0001
17	0.8937	0.7733	0.0028	<0.0001	<0.0001
21	0.7939	0.7150	0.0017	<0.0001	<0.0001

). Notably, although Glacoxan^® acted one day later than Gleba^®, it induced a stronger effect—by day 10, cultures treated with Glacoxan^® exhibited absorbance levels 1.6 times higher than those treated with Gleba^®.

When comparing the imidacloprid formulations to gibberellic acid, both exhibited stronger long-term effects. By day 21, absorbance under Glacoxan^® and Gleba^® treatments was 2.84 and 1.77 times greater, respectively, than that observed with GA, suggesting that imidacloprid may mimic or amplify hormonal signaling pathways affecting algal growth.

These findings highlight the potent and delayed growth-stimulatory effects of imidacloprid compared to classical plant hormones and underscore the need to further investigate its mode of action in non-target photosynthetic organisms.

3.3. Growth Curves of Haematococcus pluvialis, Tetradesmus sp., and Monoraphidium obtusa Under 78.2 μM of Imidacloprid Gleba^® Treatment (Assay 3)

To assess whether the growth-promoting effect of imidacloprid observed in Chlorella vulgaris was consistent across other algal taxa, we exposed three freshwater green algae—Haematococcus pluvialis, Tetradesmus sp., and Monoraphidium obtusa—to 78.2 μM of the Gleba^® commercial imidacloprid formulation for 10 days.

As shown in Figure 5, all three species exhibited significant growth stimulation, though with varying degrees of sensitivity and temporal response. The linear mixed-effects models confirmed significant effects of treatment (p < 0.001 for all species), time (p < 0.0001), and their interaction (p < 0.0001), indicating that absorbance trajectories differed across treatments over time (

Table 5. Summary of the lineal mixed models obtained for the absorbance (675 nm) of Haematococcus pluvialis, Tetradesmus sp., and Monoraphidium obtusa under 78.2 μM concentrations of imidacloprid Gleba^®. Statistically significant p-values (<0.05) are shown in bold.

		numDF	denDF	F-Value	p-Value
	(Intercept)	1	20	447.30	<0.0001
H. pluvialis	Treatment	1	5	83.99	0.0003
	Day	4	20	53.17	<0.0001
	Treatment–Day	4	20	14.51	<0.0001
	(Intercept)	1	24	2860.91	<0.0001
Tetradesmus sp.	Treatment	1	6	1406.98	<0.0001
	Day	4	24	187.59	<0.0001
	Treatment–Day	4	24	138.47	<0.0001
	(Intercept)	1	24	930.68	<0.0001
	Treatment	1	6	1013.19	0.0003
M. obtusa	Day	4	24	61.09	<0.0001
	Treatment–Day	4	24	88.54	<0.0001

).

Tetradesmus sp. and M. obtusa were the most responsive species, showing statistically significant increases in growth from day 4 onward (p < 0.0001 for both;

Table 6. Tukey contrasts between treatments and the control group for each specie. Statistically significant p-values (<0.05) are shown in bold.

Day	Haematococcus pluvialis	Tetrademus sp.	Monoraphidium obtusa
0	0.9009	0.8678	0.8910
4	0.1729	<0.0001	<0.0001
6	0.0037	<0.0001	<0.0001
7	0.0001	<0.0001	<0.0001
10	0.0003	<0.0001	<0.0001

). In contrast, H. pluvialis displayed a delayed and weaker response, with significant differences emerging only from day 6 (p = 0.0037), and a comparatively smaller effect size throughout the assay period.

By day 10, all species treated with imidacloprid exhibited significantly greater absorbance values compared to controls (p ≤ 0.0003), confirming the broad but variable sensitivity of chlorophyte algae to the Gleba^® formulation.

These findings suggest that the growth-promoting effects of imidacloprid are not limited to C. vulgaris and may extend across phylogenetically diverse freshwater algae, albeit with species-specific dynamics in onset and magnitude of response.

3.4. Growth Response of C. vulgaris to Analytical-Grade Imidacloprid (Assay 4)

To verify that the observed growth stimulation in C. vulgaris was attributable specifically to imidacloprid and not to other ingredients or adjuvants present in the commercial formulation, we conducted an independent assay using 78.2 μM of technical-grade imidacloprid for 10 days. The linear mixed-effects model revealed a significant effect of treatment (F_1,42 = 142.60, p < 0.0001), exposure time (F_3,42 = 166.54, p < 0.0001), and their interaction (F_3,42 = 97.55, p < 0.0001), indicating that both treatment and exposure duration influenced algal growth dynamics (

Table 7. Summary of the lineal mixed model obtained for the absorbance (675 nm) of C. vulgaris grown under 78.2 μM of technical-grade imidacloprid. Statistically significant p-values (<0.05) are shown in bold.

	numDF	denDF	F-Value	p-Value
(Intercept)	1	42	1106.68	<0.0001
Treatment	1	42	142.60	<0.0001
Day	3	42	166.54	<0.0001
Treatment–Day	3	42	97.55	<0.0001

). Post hoc Tukey comparisons showed no significant difference in absorbance between treated and control groups at days 0 and 3 (p = 0.6309 and p = 0.8752, respectively). However, from day 5 onwards, C. vulgaris exposed to technical-grade imidacloprid exhibited a significantly higher absorbance compared to the control (p = 0.0002 on day 5 and p < 0.0001 on day 10), suggesting a treatment-induced growth enhancement (

Table 8. Tukey contrasts between C. vulgaris grown under 7.82 μM of technical-grade imidacloprid and the control group by time. Statistically significant p-values (<0.05) are shown in bold.

Day	78.2 μM
0	0.6309
3	0.8752
5	0.0002
10	<0.0001

, Figure 6). Finally, we compared the commercial formulations under identical experimental conditions. Analytical-grade imidacloprid exhibited the most significant growth-promoting effect, which was statistically indistinguishable from that of Gleba^® (Figure 7). These results confirm that the growth-promoting effect can be attributed to imidacloprid itself, independent of formulation components.

4. Discussion

Our results demonstrate that imidacloprid stimulates C. vulgaris growth more effectively than plant hormones at equivalent concentrations, an effect we observed across different algal families.

Some other studies have assessed the impact of imidacloprid on algal growth. For instance, Malev et al. (2012) [11] found no negative effect of imidacloprid within the range of 7.6 to 255.6 mg/L on Desmodesmus subspicatus, while its transformation product (6-chloronicotinic acid) had a detrimental effect. It is noteworthy that their study only measured up to 96 h, potentially limiting their ability to observe growth induction. The same limited range of time regarding algae growth is observed in Tisler et al. (2009) [12], where, also, D. subspicatus was assayed only for 72 h.

Deng et al. (2022) [9] similarly reported that imidacloprid promoted C. vulgaris growth between 10 and 50 mg/L. In our study, we observed a similar growth-promoting effect at concentrations of 2 and 20 mg/L (7.82 and 78.2 μM, respectively). However, Deng. et al. suggested that this effect might be attributed to the utilization of imidacloprid as a carbon source by C. vulgaris. While we cannot discard this possibility, the use of alternatives carbon sources in algae is not a common characteristic and was not reported before for C. vulgaris. For instance, we have assayed the effect of imidacloprid formulations in Erlenmeyer’s with continues agitation for avoiding CO₂ limitation and the promotion of growth was constated. The removal of imidacloprid using algae has been documented in multiple studies [8,13,14,15,16] highlighting the presence of incorporation mechanisms and molecular pathways involving imidacloprid in algae, unfortunately without biotransformation products analysis. The molecular resemblance to cytokinins suggests a potential hormonal function, as cytokinin pathways have been reported in algae [17,18]. Considering that imidacloprid is a neonicotinoid, it is important to study a potential hormonal pathway linked to nicotine, particularly, the jasmonic acid pathway. Further research is necessary to elucidate the underlying mechanism.

From an ecotoxicological perspective, imidacloprid is well known for its pronounced toxicity to primary consumers such as zooplankton. Indirect stimulation of algal blooms through the suppression of grazers has been observed under low concentrations of imidacloprid in mesocosm studies [19]. Thus, trophic cascade effects may represent the primary ecological mechanism through which imidacloprid alters community dynamics in freshwater systems [20]. Nevertheless, our findings suggest that direct stimulation of algal growth by imidacloprid could also play a role—particularly in high-contamination scenarios—adding an additional layer of complexity to its ecological impacts.

5. Conclusions

High concentrations of imidacloprid promote algal growth, potentially heightening aquatic toxicological risks under worst-case scenarios. As this effect exceeds that of certain plant hormones, further research is needed to uncover the underlying molecular mechanisms.