Environmental concentrations of metformin exposure affect aggressive behavior in the Siamese fighting fish, Betta splendens

Abstract

Metformin, the medicine most commonly prescribed for treatment of Type II diabetes, is among the most abundant pharmaceuticals being introduced into the environment. Pharmaceuticals are increasingly found in wastewater and surface waters around the world, often due to incomplete metabolism in humans and subsequent excretion in human waste. Risk analyses and exposure studies have raised concerns about potential negative impacts of pharmaceuticals at current environmental levels. Results of the present study indicate that metformin at concentrations in the range of what has been documented in freshwater systems and waste-water effluent (40 μg/L) affects aggressive behavior in adult male Betta splendens. Subjects exhibited less aggression toward a male dummy stimulus after four weeks exposure to metformin-treated water when compared to behavior measured immediately prior to their exposure, and in comparison to a separate cohort of un-exposed control fish. This effect persisted after 20 weeks exposure as well. Subjects exposed to metformin at a concentration twice that currently observed in nature (80 μg/L) exhibited an even more substantial reduction in aggressive behaviors compared to controls and pre-exposure measurements than those observed in the low-dose treatment group. Such changes in behavior have the potential to affect male fitness and possibly impact the health of natural populations of aquatic organisms exposed to the drug.

Fig 1

(A) Arrangement of 9.5 L (30 X 15 X 15 cm) aquaria used for all behavioral assays throughout the five-month study period. The subject in its home tank was placed between a pair of aquaria each housing an adjustable motorized belt and pulley system. The male dummy stimulus was attached to the pulley system situated in the aquarium to the right side of the subject’s home tank for all behavioral assays. When the apparatus was turned on, the dummy fish began ‘pacing’ back and forth along the glass facing the subject tank. An iPad was used to record the subject’s behavior for 5 min following the removal of opaque barriers blocking his view of the dummy in the neighboring tank. (B) Image used to create the 6.3 cm total length male dummy used in all behavioral assays.

Fig 2. Means (±SE) for behaviors directed toward the male dummy stimulus.

The amount of time (sec) males in the low-dose (LD; 40 μg/L metformin), high-dose (HD; 80 μg/L metformin), and the control groups (C; no metformin exposure) spent gill flaring in each of three sequences of identical behavioral assays (pre-exposure, 4 weeks exposure, and 20 weeks exposure). Letter designations (a and b) correspond with significant p-values presented in Table 1 and indicate significant differences among the low-dose, high-dose, and control group means within a given time period of testing (e.g. comparing the HD vs LD vs C group responses after four weeks exposure) (See Table 1 for statistical results).

Fig 3. Means (±SE) for behaviors directed toward the male dummy stimulus.

The amount of time (sec) males in the LD, HD, and C groups spent fin spreading in each of the three sequences of behavioral assays (PE, 4-WK, and 20-WK). The letter designation (c) corresponds with significant p-values presented in Table 1 and indicate significant differences among the low-dose, high-dose, and control group means within the given time period of testing (See Table 1 for statistical results).

Fig 4. Means (±SE) for behaviors directed toward the male dummy stimulus.

Number of tail beats subjects in the LD, HD, and C groups directed towards the male dummy in each of the three sequences of behavioral assays (PE, 4-WK, and 20-WK). The letter designation (d) corresponds with significant p-values presented in Table 1 and indicate significant differences among the low-dose, high-dose, and control group means within the given time period of testing (See Table 1 for statistical results).