Toxicity and Safety Assessment of Key Pesticides Used in Rice Fields on Rice Flower Carp (Procypris merus)

Abstract

Integrated rice–fish farming, crucial for sustainable agriculture, relies on the judicious use of pesticide. This study evaluates the toxicity of six common rice-field pesticides on Procypris merus (rice flower carp), a key species in these systems. We conducted acute and chronic toxicity tests, assessing survival, growth, oxidative stress (SOD, CAT, MDA, 8-OHdG), and neurotoxicity (AChE). Results revealed a spectrum of toxicity: abamectin and trifloxystrobin were highly toxic; pretilachlor was moderately so; and glufosinate-ammonium, triflumezopyrim, and thiazole zinc were low. Notably, triflumezopyrim induced significant oxidative stress and DNA damage, while all three low-toxicity pesticides inhibited AChE activity, indicating potential neurotoxicity. Despite these effects, all observed toxicities were reversible within 7–14 days. Considering that the tested concentrations exceeded typical field application rates, glufosinate-ammonium, triflumezopyrim, and thiazole zinc are deemed relatively safe for P. merus at recommended dosages. Our findings provide critical insights for optimizing pesticide selection in rice–fish farming, balancing pest control with ecological safety, thereby informing sustainable agricultural practices.

1. Introduction

Integrated rice–fish farming is a widespread agricultural practice in China [1], designed to simultaneously optimize pest and disease control while ensuring the safety of aquatic organisms within rice paddy ecosystems [2]. The choice of pesticides significantly impacts the economic and environmental sustainability of this model. Despite existing toxicity evaluations of pesticides on various model organisms, research on their effects on farmed aquatic species specifically within these integrated systems remains insufficient [3]. Procypris merus (rice flower carp), a fish species indigenous to Guilin, Guangxi, China, is frequently used in integrated rice–fish farming systems due to its high adaptability [4,5].

Avermectin demonstrates excellent efficacy against Chilo suppressalis and Cnaphalocrocis medinalis [6], but shows toxic effects on aquatic organisms including Danio rerio [7], Oreochromis niloticus [8], and Cyprinus carpio [9]. Similarly, triflumezopyrim acts by binding to the orthosteric site of nAChRs, inducing insect lethargy and poisoning [10]. While effective against rice planthoppers [11], triflumezopyrim exhibits toxicity to aquatic species like Labeo rohita, with chronic exposure causing hepatic histopathological alterations and impaired antioxidant capacity [12].

Pretilachlor, a chloroacetamide herbicide [13], disrupts protein synthesis in plants, progressively affecting photosynthesis and respiration until plant death [14]. Studies have demonstrated PR’s aquatic toxicity. Soni and Verma [15] observed significantly reduced feeding behavior and increased burst swimming in Clarias batrachus, along with respiratory impairment and reproductive toxicity. PR also induces endocrine disruption, apoptosis, oxidative stress, and immunotoxicity in D. rerio embryos [16], causing developmental abnormalities in liver, skeleton, and swim bladder [17]. Glufosinate-ammonium, a broad-spectrum organophosphorus herbicide [18], competitively inhibits glutamine synthase (GS), rapidly killing plant cells [19,20]. As GS exists in both plants and animals [21], GLA adversely affects aquatic organisms, inducing hepatic oxidative stress, inflammation, and apoptosis in D. rerio [22], along with immunotoxicity and potential reproductive toxicity in embryos [23].

Trifloxystrobin, a widely used strobilurin fungicide in rice, vegetables, fruits, and soybeans [24], causes developmental abnormalities in the liver, ovary, and heart of D. rerio [25], induces mitochondrial dysfunction and apoptosis [26], and shows high embryo toxicity [27,28]. Zinc thiazole, a China-developed organic zinc fungicide with low mammalian toxicity [29], effectively controls bacterial diseases like rice bacterial blight and bacterial leaf streak [30]. Limited studies report its aquatic toxicity: Tong et al. [31] found high acute toxicity to Daphnia magna, with chronic exposure inhibiting growth and causing reproductive impairment.

This study assesses the toxicity of six commonly used rice paddy pesticides on P. merus, with a focus on their effects on growth, physiological parameters, and oxidative stress responses, to establish a scientific foundation for safe pesticide use in integrated rice–fish farming systems. Furthermore, this research addresses a critical gap in understanding the ecological consequences of pesticide use in integrated aquaculture systems, offering essential data to inform policy-making, environmental risk assessments, and the development of sustainable agricultural practices that balance productivity with ecological safety.

2. Materials and Methods

2.1. Test Organisms, Materials, and Reagents

Procypris merus (average body length: 4.99 ± 0.15 cm) were purchased from Qian’s Aquatic Products Trading Department, Foshan City, Guangdong Province. Fish were acclimated in laboratory aquariums (45 cm × 17 cm × 30 cm) for more than 7 days under controlled conditions (24 ± 1 °C, pH 6.81 ± 0.23, water hardness 80 mg/L as CaCO₃, dissolved oxygen > 5 mg/L). Dechlorinated tap water, aerated for 3 days, was used. Only healthy individuals with <1% natural mortality were selected. Fish were fed once daily with commercial fish food (1% body weight) during acclimation and fasted 24 h before experiments.

Acetone (analytical grade) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Abamectin (95% technical grade) was purchased from Shanghai Meiruier Biochemical Technology Co., Ltd. (Shanghai, China). Trifloxystrobin (98% technical grade) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Glufosinate (97% technical grade) was purchased from Shanghai Meiruier Biochemical Technology Co., Ltd. (Shanghai, China). Pretilachlor (95% technical grade) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Thiazole zinc (40% suspension concentrate) was purchased from Zhejiang Xinnong Chemical Co., Ltd. (Hangzhou, China). Triflumezopyrim (10% suspension concentrate) was purchased from Corteva Agriscience LLC (Indianapolis, IN, USA).

2.2. Acute Toxicity Testing

A 96 h semi-static test was conducted, with test solutions replaced every 24 h. Based on preliminary tests, six concentration gradients (equal logarithmic intervals) were established between the highest survival and lowest mortality concentrations for each pesticide. Solvent (acetone) and blank controls were included. For pesticides showing no mortality at 100 mg/L, low toxicity was assumed.

Ten P. merus specimens were randomly assigned to 20 L aquariums containing 5 L of test solution (three replicates per concentration). Mortality was recorded at 24, 48, 72, and 96 h. Test solutions were replaced daily. Dead fish (no response after 30 s of gentle prodding) were promptly removed.

2.3. Chronic Toxicity Testing

Chronic toxicity tests were performed on low-toxicity pesticides identified in acute toxicity tests. Concentrations were set at 1/10 and 1/100 of the 96 h LC₅₀ values and compared with recommended field application rates (

Table 1. Concentration settings for chronic toxicity testing (mg/L).

Pesticide	Field Relevant	1/10 LC50	1/100 LC50
Trflumezopyrim	3.26	1.93	0.19
Glufosinate-Ammonium	7.50	10.00	1.00
Zinc Thiazole	8.36	6.79	0.68

).

Thirty P. merus specimens were assigned to each treatment group (three replicates). Fish were exposed to pesticides for 7 days, followed by a 14-day recovery period in clean water. On days 0, 7, 14, and 21, six fish were randomly sampled from each replicate for body length, body weight, and liver weight measurements. The following formulae were used for calculations:

SGR (Specific growth rate, % g/d) = (lnW_t − lnW₀)/t × 100%

(1)

CF (Condition factor) = W_t/L_t × 100%

(2)

HSI (Hepatic steatosis index, %) = W_h/W_t × 100%

(3)

In the formulae, t represents the number of farming days (d); W_t denotes the final body weight (g); W₀ represents the initial body weight (g); L_t denotes the final body length (cm); and W_h represents the hepatopancreas weight (g).

Six P. merus specimens were randomly selected. Hepatopancreas tissue (0.20 g) was homogenized in 0.9% saline solution (1:10 w/v) at 4 °C and centrifuged (10 °C, 2500 r/min), and the supernatant was used for analysis. Superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and acetylcholinesterase (AChE) levels were measured using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). 8-hydroxy-2′-deoxyguanosine (8-OHdG) content was determined using a Fish 8-Hydroxydeoxyguanosine (8-OHdG) ELISA Kit.

2.4. Statistical Analysis

Probit regression (SPSS 26.0) was used to calculate LC₅₀ values and 95% confidence limits. Toxicity levels were classified according to Chinese standard GB/T 31270-2014 [32].

Survival analysis was performed using the Kaplan–Meier method and Log-rank test. One-way ANOVA followed by Duncan’s multiple comparison test was used to analyze treatment differences. Graphs were generated using GraphPad Prism 10.1.2.

3. Results

3.1. Acute Toxicity of Rice Field Pesticides to P. merus

Abamectin:

Abamectin demonstrated high acute toxicity to P. merus, with a concentration-dependent decrease in survival rates (Figure 1A). After 96 h of exposure, significant reductions in survival were observed at concentrations of 2.82 × 10⁻² mg/L, 3.36 × 10⁻² mg/L, and 4.40 × 10⁻² mg/L compared to the control group (2.82 × 10⁻² mg/L: df = 6, χ² = 13.328, p < 0.001; 3.36 × 10⁻² mg/L: df = 6, χ² = 35.786, p < 0.001; 4.40 × 10⁻² mg/L: df = 6, χ² = 64.454, p < 0.001). The 96 h LC₅₀ value was 0.30 × 10⁻¹ mg/L (

Table 2. Acute toxicity of rice-field pesticides to Procypris merus. * indicates a significant correlation at the 0.05 level, and ** indicates a highly significant correlation at the 0.001 level.

Type	Pesticides	Treatment (h)	Regression Equation	Correlation R2	LC50 (mg/L)	95% Confidence Interval	Toxicity Grade
Insecticide	Avermectin
		24	-	-	-	-	-
		48	y = 14.14x + 21.08	0.98 **	0.32 × 10−1	0.30 × 10−1–0.36 × 10−1	Extreme Toxicity
		72	y = 17.55x + 26.54	0.98 **	0.31 × 10−1	0.28 × 10−1–0.33 × 10−1	Extreme Toxicity
		96	y = 17.07x + 25.97	0.99 **	0.30 × 10−1	0.28 × 10−1–0.33 × 10−1	Extreme Toxicity
	Trflumezopyrim
		24	-	-	10.00 < LC50 < 29.24	-	Low Toxicity
		48	-	-	10.00 < LC50 < 29.24	-	Low Toxicity
		72	-	-	10.00 < LC50 < 29.24	-	Low Toxicity
		96	-	-	10.00 < LC50 < 29.24	-	Low Toxicity
Herbicide	Pretilachlor
		24	-	-	-	-	-
		48	y = 12.16x − 10.31	0.75	7.04	-	Moderate Toxicity
		72	y = 14.59x − 9.61	0.98 **	4.56	4.23–4.15	Moderate Toxicity
		96	y = 16.01x − 9.29	0.99 **	3.80	3.54–4.08	Moderate Toxicity
	Glufosinate-ammonium
		24	-	-	LC50 > 100.00	-	Low Toxicity
		48	-	-	LC50 > 100.00	-	Low Toxicity
		72	-	-	LC50 > 100.00	-	Low Toxicity
		96	-	-	LC50 > 100.00	-	Low Toxicity
Fungicide	Trifloxystrobin
		24	-	-	LC50 < 0.10	-	Extreme Toxicity
		48	-	-	LC50 < 0.10	-	Extreme Toxicity
		72	-	-	LC50 < 0.10	-	Extreme Toxicity
		96	-	-	LC50 < 0.10	-	Extreme Toxicity
	Zinc Thiazole
		24	y = 1.65x − 4.29	0.98 *	390.89	-	Low Toxicity
		48	y = 3.27x − 7.64	0.99 **	215.69	148.52–339.08	Low Toxicity
		72	y = 3.88x − 7.40	1.00 **	80.36	57.24–117.33	Low Toxicity
		96	y = 2.67x − 4.88	0.99 **	67.89	45.66–109.18	Low Toxicity

), indicating extreme toxicity.

Triflumezopyrim:

In contrast, triflumezopyrim displayed low toxicity to P. merus, with no observed mortality at 10.00 mg/L after 96 h, and 100% mortality at 29.24 mg/L (Figure 1B). The 96 h LC₅₀ was therefore determined to be between 10.00 and 29.24 mg/L (Table 2), classifying it as having low toxicity.

Pretilachlor:

Pretilachlor exhibited moderate toxicity. After 72 h, survival rates decreased significantly by 80.00%, 53.33%, and 33.33% at concentrations of 3.87 mg/L, 4.40 mg/L, and 5.00 mg/L, respectively, compared to the control (3.87 mg/L: df = 5, χ² = 21.386, p < 0.001; 4.40 mg/L: df = 5, χ² = 37.579, p < 0.001; 5.00 mg/L: df = 5, χ² = 62.345, p < 0.001) (Figure 1C). The 96 h LC₅₀ was 3.80 mg/L (Table 2), indicating moderate toxicity.

Glufosinate-ammonium:

Glufosinate-ammonium exhibited low toxicity, with no mortality observed after 96 h at 100.00 mg/L (Figure 1D). This indicates a 96 h LC₅₀ greater than 100.00 mg/L (Table 2).

Trifloxystrobin:

Trifloxystrobin showed high toxicity; complete mortality occurred at 0.1 mg/L after 96 h (Figure 1E), indicating a 96 h LC₅₀ less than 0.10 mg/L (Table 2).

Thiazole zinc:

Thiazole zinc exhibited moderate toxicity. Significant reductions in survival were observed after 96 h at 47.29 mg/L, 100.00 mg/L, 211.47 mg/L, and 447.21 mg/L (47.29 mg/L: df = 7, χ² = 10.442, p < 0.001; 100.00 mg/L: df = 7, χ² = 21.329, p < 0.001; 211.47 mg/L: df = 7, χ² = 58.764, p < 0.001; 447.21 mg/L: df = 7, χ² = 66.381, p < 0.001) (Figure 1F). Complete mortality was observed at 211.47 mg/L and 447.21 mg/L after 72 h. The 96 h LC₅₀ was 67.89 mg/L (Table 2).

Summary of toxicity:

Among the six pesticides tested, triflumezopyrim, glufosinate-ammonium, and thiazole zinc exhibited low toxicity to P. merus. Pretilachlor showed moderate toxicity, while abamectin and trifloxystrobin were highly toxic.

3.2. Effects of Rice-Field Chemical Pesticides on the Growth of P. merus

Triflumezopyrim:

Exposure to triflumezopyrim significantly inhibited the growth of P. merus after 7 days (df = 3, F = 41.540, p < 0.001) (Figure 2A). The specific growth rate (SGR) was lowest in the 3.26 mg/L group, showing a 19.34% reduction compared to the control. Significant reductions in SGR were also observed at 0.19 mg/L (5.62%) and 1.93 mg/L (16.02%). After a 14-day recovery period in clean water, no significant differences in SGR were found among the groups (df = 3, F = 0.217, p = 0.884) (Figure 2A). Although no significant differences in body weight were observed after 7 days of exposure (df = 3, F = 1.877, p = 0.139), significant differences emerged after the 14-day recovery (df = 3, F = 5.014, p = 0.003). Body weights in the 0.19 mg/L, 1.93 mg/L, and 3.26 mg/L groups were reduced by 0.04 g, 0.13 g, and 0.22 g, respectively, compared to the control (Figure 2B).

Thiazole zinc:

Thiazole zinc significantly inhibited growth after 7 days (df = 2, F = 524.675, p < 0.001) (Figure 2C). SGR reductions of 38.12% and 42.57% were observed at 0.68 mg/L and 6.79 mg/L, respectively (Figure 2C). After a 14-day recovery, no significant differences in SGR were detected (df = 2, F = 0.060, p = 0.942) (Figure 2C). Significant body weight differences were observed after 7 days (df = 2, F = 5.753, p = 0.005), with reductions of 0.13 g and 0.07 g in the 0.68 mg/L and 6.79 mg/L groups, respectively (Figure 2D). These differences persisted after the 14-day recovery (df = 2, F = 6.359, p = 0.003), with reductions of 0.21 g and 0.11 g (Figure 2D).

Glufosinate-ammonium:

Glufosinate-ammonium significantly reduced SGR after 7 days (df = 2, F = 107.430, p < 0.001) (Figure 2E). SGR reductions of 17.40% and 30.71% were recorded at 1 mg/L and 10 mg/L, respectively (Figure 2E). After a 14-day recovery, no significant differences in SGR were found (df = 2, F = 0.148, p = 0.863) (Figure 2E). Although body weight differences were not significant after 7 days (df = 2, F = 1.857, p = 0.164), significant differences emerged after the recovery period (df = 2, F = 3.982, p = 0.023), with body weights reduced by 0.12 g and 0.21 g in the 1.00 mg/L and 10.00 mg/L groups, respectively (Figure 2F).

• Hepatosomatic index (HSI) and condition factor (CF):

After 7 days of exposure, the hepatosomatic index (HSI) of P. merus was significantly reduced by all three pesticides (triflumezopyrim: df = 3, F = 3.649, p = 0.017; thiazole zinc: df = 2, F = 12.878, p < 0.001; glufosinate-ammonium: df = 2, F = 3.877, p = 0.028) (

Table 3. Effects of three pesticides used in rice field on morphological indices of P. merus. Data are mean ± SE. Different lowercase letters indicate significant differences between treatment groups at the same time point (p < 0.05); the same below.

Pesticide	Treatment	Concentration (mg/L)	Condition Factor (g/cm)	Hepatosomatic Index (%)
Trflumezopyrim	0 d	0.00	40.39 ± 0.78	3.54 ± 0.16
		0.19	40.60 ± 1.03	3.56 ± 0.24
		1.93	38.62 ± 0.72	3.59 ± 0.20
		3.26	39.06 ± 0.70	3.58 ± 0.22
	7 d	0.00	42.31 ± 0.39	3.56 ± 0.04 a
		0.19	42.49 ± 0.33	3.52 ± 0.04 a
		1.93	41.48 ± 0.43	3.45 ± 0.05 ab
		3.26	41.43 ± 0.30	3.38 ± 0.04 b
	14 d	0.00	48.12 ± 0.25	3.55 ± 0.05
		0.19	47.97 ± 0.23	3.56 ± 0.03
		1.93	47.47 ± 0.25	3.54 ± 0.03
		3.26	47.90 ± 0.25	3.55 ± 0.04
	21 d	0.00	55.78 ± 0.37	3.61 ± 0.02
		0.19	55.78 ± 0.40	3.57 ± 0.03
		1.93	54.83 ± 0.37	3.58 ± 0.03
		3.26	55.39 ± 0.34	3.57 ± 0.04
Glufosinate-Ammonium	0 d	0.00	40.39 ± 0.78	3.54 ± 0.16
		1.00	39.12 ± 0.68	3.53 ± 0.15
		10.00	38.83 ± 1.07	3.54 ± 0.24
	7 d	0.00	42.31 ± 0.39	3.56 ± 0.04 a
		1.00	41.62 ± 0.39	3.32 ± 0.04 b
		10.00	41.47 ± 0.17	3.07 ± 0.11 c
	14 d	0.00	47.59 ± 0.39	3.55 ± 0.05 a
		1.00	47.22 ± 0.27	3.43 ± 0.07 ab
		10.00	46.85 ± 0.41	3.31 ± 0.06 b
	21 d	0.00	55.78 ± 0.37	3.61 ± 0.02
		1.00	54.91 ± 0.46	3.59 ± 0.04
		10.00	53.72 ± 0.31	3.52 ± 0.04
Zinc Thiazole	0 d	0.00	40.39 ± 0.78	3.54 ± 0.16
		0.68	41.33 ± 1.14	3.46 ± 0.18
		6.79	39.78 ± 1.23	3.53 ± 0.26
	7 d	0.00	42.31 ± 0.39	3.56 ± 0.04 a
		0.68	42.12 ± 0.28	3.38 ± 0.09 a
		6.79	41.51 ± 0.22	2.99 ± 0.10 b
	14 d	0.00	47.59 ± 0.39	3.55 ± 0.05
		0.68	47.12 ± 0.28	3.54 ± 0.03
		6.79	46.66 ± 0.29	3.56 ± 0.03
	21 d	0.00	55.78 ± 0.37	3.61 ± 0.02
		0.68	55.03 ± 0.29	3.58 ± 0.03
		6.79	55.10 ± 0.24	3.59 ± 0.04

). The lowest HSI was observed in the 3.26 mg/L triflumezopyrim group. Exposure to 6.79 mg/L thiazole zinc resulted in a 16.01% reduction in HSI. Glufosinate-ammonium at 1.00 mg/L and 10.00 mg/L led to HSI reductions of 6.74% and 13.76%, respectively. After a 7-day recovery, no significant differences in HSI were observed (triflumezopyrim: df = 2, F = 107.430, p < 0.001; thiazole zinc: df = 2, F = 107.430, p < 0.001; glufosinate-ammonium: df = 2, F = 9.737, p < 0.001) (Table 3). No significant effects on the condition factor (CF) were observed for any pesticide (Table 3).

3.3. Effects of Rice-Field Pesticides on the Antioxidant Enzyme Activity of P. merus

Superoxide Dismutase (SOD) Activity:

Exposure to triflumezopyrim for 7 days significantly inhibited superoxide dismutase (SOD) activity in the hepatopancreas of P. merus (df = 3, F = 22.300, p < 0.001) (Figure 3A). SOD activity decreased in a concentration-dependent manner across the range of 0.19 to 3.26 mg/L. After a 7-day recovery period, SOD activity returned to levels comparable to the control, with no significant differences among groups (df = 3, F = 0.865, p = 0.498) (Figure 3A). In contrast, neither thiazole zinc (df = 2, F = 2.689, p = 0.147) nor glufosinate-ammonium (df = 2, F = 0.205, p = 0.820) significantly affected SOD activity (Figure 3B,C).

Exposure to triflumezopyrim for 7 days significantly inhibited catalase (CAT) activity in the hepatopancreas of P. merus (df = 3, F = 5.386, p = 0.025) (Figure 4A). Following a 7-day recovery period, no significant differences in CAT activity were observed between the treatment groups and the control group (df = 3, F = 1.471, p = 0.294) (Figure 4A).

Similarly, neither thiazole zinc (df = 2, F = 1.415, p = 0.314) nor glufosinate-ammonium (df = 2, F = 0.751, p = 0.512) significantly affected CAT activity (Figure 4B,C).

3.4. Effects of Rice-Field Pesticides on Malondialdehyde (MDA) Content in P. merus

Exposure to triflumezopyrim for 7 days significantly altered malondialdehyde (MDA) content in the hepatopancreas of P. merus (df = 3, F = 4.708, p = 0.035) (Figure 5A). Specifically, the 3.26 mg/L triflumezopyrim treatment significantly increased MDA content compared to the control. After a 7-day recovery, no significant differences in MDA were observed (df = 3, F = 1.175, p = 0.378) (Figure 5A). Neither thiazole zinc (df = 2, F = 0.925, p = 0.447) nor glufosinate-ammonium (df = 2, F = 0.533, p = 0.612) significantly affected MDA content (Figure 5B,C).

3.5. Effects of Paddy-Field Pesticides on Acetylcholinesterase (AChE) Activity in P. merus

Exposure to triflumezopyrim, thiazole zinc, and glufosinate-ammonium for 7 days significantly inhibited acetylcholinesterase (AChE) activity in P. merus (triflumezopyrim: df = 3, F = 18.059, p = 0.001; thiazole zinc: df = 2, F = 18.065, p = 0.003; glufosinate-ammonium: df = 2, F = 9.500, p = 0.014) (Figure 6). Specifically, 1.93 mg/L and 3.26 mg/L triflumezopyrim, 6.79 mg/L thiazole zinc, and 1.00 mg/L and 10.00 mg/L glufosinate-ammonium significantly reduced AChE activity compared to the control. After a 7-day recovery, no significant differences in AChE activity were observed (triflumezopyrim: df = 2, F = 1.643, p = 0.270; thiazole zinc: df = 2, F = 0.223, p = 0.807; glufosinate-ammonium: df = 2, F = 0.695, p = 0.535) (Figure 6).

3.6. Effects of Paddy Field Pesticides on 8-Hydroxy-2′-Deoxyguanosine (8-OHdG) Content in P. merus

Exposure to triflumezopyrim for 7 days significantly increased 8-hydroxy-2′-deoxyguanosine (8-OHdG) content in the hepatopancreas of P. merus (df = 3, F = 8.656, p = 0.007) (Figure 7A), indicating oxidative DNA damage. After a 7-day recovery, no significant differences in 8-OHdG were observed (df = 3, F = 0.699, p = 0.579) (Figure 7A). Neither thiazole zinc (df = 2, F = 1.072, p = 0.400) nor glufosinate-ammonium (df = 2, F = 2.342, p = 0.177) significantly affected 8-OHdG content (Figure 7B,C).

4. Discussion

4.1. Toxicity Analysis of Paddy-Field Pesticides on P. merus

This study aimed to identify pesticides with low risk to P. merus in integrated rice–fish farming by assessing the toxicity of six common rice paddy pesticides. Results categorized glufosinate-ammonium, triflumezopyrim, and thiazole zinc as low-toxicity, pretilachlor as moderately toxic, and abamectin and trifloxystrobin as highly toxic. P. merus, like other cyprinids, is known to be sensitive to pollutants [33]. The low toxicity of glufosinate-ammonium is consistent with findings in Eriocheir sinensis [34]. Glufosinate-ammonium (GLA), a broad-spectrum herbicide [18,35], inhibits glutamine synthase (GS) [19,20], an enzyme present in both plants and animals [21]. Although primarily herbicidal, GLA can induce oxidative stress, inflammation, and apoptosis in fish liver [22] and can exhibit immunotoxicity and reproductive toxicity in zebrafish embryos [23].

Triflumezopyrim, a nicotinic acetylcholine receptor (nAChR) antagonist [36], can impact liver structure and antioxidant capacity in fish like L. rohita [12]. Thiazole zinc, a fungicide [29], has limited toxicity data on aquatic organisms, but has demonstrated high acute toxicity to D. magna and adverse effects on growth and reproduction with prolonged exposures [31].

Pretilachlor’s moderate toxicity to P. merus is in agreement with findings in C. carpio [37]. This chloroacetamide herbicide disrupts protein synthesis [13,14] and can induce endocrine disruption, apoptosis, oxidative stress, and immunotoxicity in fish [17]. Soni et al. [38] found that PR significantly reduces the feeding behavior of C. batrachus, significantly increases its explosive swimming behavior, adversely affects the respiratory process of the fish, and is reproductively toxic.

Abamectin and trifloxystrobin were found to be highly toxic. Abamectin induces paralysis by stimulating GABA release [39] and has shown high toxicity in zebrafish [7], tilapia [8], and carp [9]. Some studies found that the acute-toxicity 96 h LC₅₀ of abamectin to Oncorhynchus mykiss was 0.003 mg/L [40] and to zebrafish (D. rerio) was 59 μg/L at 96 h LC₅₀ [41]; in the current study, the 96 h LC₅₀ was 0.030 mg/L, which is highly toxic. However, the sensitivity of P. merus to avermectin was lower than that of O. mykiss. Trifloxystrobin, an ATP synthesis inhibitor, causes mitochondrial dysfunction [42] and developmental abnormalities [25,26]. Studies in grass carp and zebrafish further corroborate its high toxicity [43].

This study suggests that triflumezopyrim, glufosinate-ammonium, and thiazole zinc represent lower-risk pesticides for integrated rice–fish systems, provided that application dosages are within recommended limits.

4.2. Effects of Paddy Field Pesticides on the Antioxidant System of P. merus

Exposure to pesticides induces the production of reactive oxygen species (ROS), leading to oxidative stress [44]. Superoxide dismutase (SOD) and catalase (CAT) are critical antioxidant enzymes [6]. However, triflumezopyrim significantly reduced both SOD and CAT activities while increasing malondialdehyde (MDA) levels, suggesting an overload of ROS and consequent oxidative damage. This phenomenon may be attributed to the limited ROS-scavenging capacity of antioxidant enzymes. With increasing pesticide concentrations or prolonged exposure durations, the antioxidant defense system becomes overwhelmed and fails to prevent oxidative damage in the hepatopancreas of test organisms [45]. Consequently, this leads to the suppression of enzymatic activities and triggers lipid peroxidation, as evidenced by elevated MDA production [28]. This inhibition likely reflects overwhelmed antioxidant defenses, consistent with other studies [6,46]. Huang et al. [47] found that glyphosate at 20 mg/L for 96 h caused damage to antioxidant and immune enzymes and tissue structure of P. clarkii, disrupting basic physiological functions and behaviors. Importantly, the observed damage was reversible, indicating that no irreversible harm occurred. Further investigation into repair mechanisms is warranted.

4.3. Effects of Rice-Field Pesticides on DNA Oxidative Damage in P. merus

Oxidative DNA damage, as measured by 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels, is linked to ROS accumulation [48]. Triflumezopyrim significantly increased 8-OHdG content, suggesting that oxidative stress led to DNA damage. This finding is consistent with studies on other pesticides. The fungicide flumioxazin significantly induced 8-OHdG production in D. rerio embryos [49]. Avermectin exposure led to an increase in 8-OHdG content in E. sinensis, which in turn triggered oxidative DNA damage and exhibited some genotoxicity [50]. Dose-dependent elevation in 8-OHdG activity in the brain tissues of L. rohita during triflumezopyrim exposure, which had a pronounced impact on 8-OHdG levels [51]. The recovery of 8-OHdG levels indicates that DNA damage was reversible.

4.4. Effects of Rice-Field Pesticides on Neural Transmission in P. merus

Acetylcholinesterase (AChE) inhibition is a well-established marker of neurotoxicity [52]. In our study, exposure to triflumezopyrim, glufosinate-ammonium, and thiazole zinc resulted in significant AChE inhibition, which may lead to neurotoxic effects. The insecticide fipronil significantly inhibited AChE activity in D. rerio, resulting in a large accumulation of acetylcholine, which triggered avoidance behavior in zebrafish, significantly reducing their swimming speed and distance traveled [53]. Capkin et al. [54] found that the insecticide thiophanate significantly inhibited AChE activity in the liver of O. mykiss, with recovery taking 21 d to normalize. The reversibility of AChE inhibition suggests that the neurotoxic effects were transient.

4.5. Effects of Paddy-Field Pesticides on the Growth of P. merus

Triflumezopyrim, glufosinate-ammonium, and thiazole zinc inhibited the growth of P. merus, likely due to reduced AChE activity, impacting feeding behavior. Zhu et al. [55] found that the inhibition of AChE activity may block neurotransmission and affect feeding, leading to the paralysis and death of the organism. In the normal state of an organism, the ratio of weight of tissues to body weight is relatively constant, and, after stress by pollutants, the weight of damaged organs may be abnormal; hepatopancreatic index is often used to assess the degree of hepatopancreatic damage [56]. Dogan et al. [57] found that O. mykiss exposed to the pesticide thiamethoxam showed tissue hypertrophy and an elevated hepatopancreatic index. In the present study, triflumezopyrim significantly reduced the hepatopancreatic index in P. merus after 7 days of stress, as did glufosinate ammonium and thiazolium zinc, and it was hypothesized that P. merus suffered from hepatopancreatic atrophy or other degenerative alterations after exposure to stress [58]. Additionally, reductions in the hepatosomatic index (HSI) were observed, suggesting potential hepatopancreatic atrophy. Nevertheless, the observed growth impairment was reversible, indicating that no permanent harm occurred.

5. Conclusions

This study provided a comprehensive evaluation of the toxic effects of six rice-paddy pesticides on Procypris merus. Notably, glufosinate-ammonium, triflumezopyrim, and thiazole zinc demonstrated low toxicity, whereas pretilachlor was moderately toxic and abamectin and trifloxystrobin exhibited high toxicity.

Mechanistically, triflumezopyrim induced significant oxidative stress in P. merus, as evidenced by reduced superoxide dismutase (SOD) and catalase (CAT) activities, and increased levels of MDA and 8-OHdG. Furthermore, all three low-toxicity pesticides (triflumezopyrim, glufosinate-ammonium, and thiazole zinc) significantly inhibited AChE activity, indicating potential neurotoxic effects, and they also impaired growth, as reflected by reduced specific growth rate (SGR) and overall performance.

Importantly, all observed toxic effects were reversible within 7 to 14 days, suggesting that no irreversible harm occurred. Given that the high-concentration treatments in this study exceeded recommended field application rates, triflumezopyrim, glufosinate-ammonium, and thiazole zinc are considered relatively safe for P. merus at recommended dosages, making them suitable for integrated rice–fish farming systems. Conversely, abamectin, pretilachlor, and trifloxystrobin are not recommended for such systems. These findings corroborate earlier studies by demonstrating that, while some pesticides cause reversible toxic effects, others (e.g., abamectin, pretilachlor, trifloxystrobin) pose significant risks under high-dose exposures. Beyond their immediate implications for aquaculture, these findings contribute to a broader understanding of pesticide interactions with aquatic ecosystems. By integrating toxicological data into environmental risk assessments and sustainable farming policies, this study underscores the need for scientifically informed pesticide management strategies that protect both agricultural productivity and ecological integrity.