Development of Daphnia Magna Under Exposure to Ampicillin

The test organism was Daphnia magna Straus, purchased from MicroBio Tests Inc. Belgium. The animal has been fed with green alga Chlorella sp. and maintained in the laboratory conditions of 22 ± 1°C, dim light and light dark cycle of 14h:10h, for many generations. The D. magna was reared in ISO medium consisted of NaHCO₃, CaCl₂, MgSO₄ and KCl while the green alga was cultivated in Z8 medium under continuous aeration [5].

Before to the experiment, thirty active adolescent female daphnids were selected and incubated in a 500 mL beaker containing 400 mL ISO medium for approximately 2–3 weeks. During this period, the daphnids were fed with ad libitum alga. The offspring (<24 h) from the second to third clutch of these female Daphnia were chosen randomly for the test. The chemical ampicillin anhydrous C₁₆H₁₉N₃O₄S (99% from Sigma Aldrich, St. Louis, Mo., USA) was used for the chronic exposure to D. magna. Because ampicillin rapidly loses activity when stored above a pH of 7.0 [3, 10, 19], the antibiotic solution was dissolved in acetate buffers which pH value was approximately 4 [3, 10]. The concentration of this stock was 5 mg/L and stored at the temperature of 4°C.

The neonates (15 individuals per treatment) were individually incubated in 50 mL glass cups containing 20 mL ISO medium solely considered as control. For the antibiotic exposures, D. magna was incubated in medium containing 5, 50 and 250 µg ampicillin/L (abbreviated as 5, 50 and 250 ppb). The animals were incubated in the laboratory conditions as mentioned above. The test media were totally renewed 3 times a week, and the animals were fed with Chlorella sp. at the concentration of 1 mg/L per d. All incubations lasted for 3 weeks. The fitness parameters of Daphnia daily observed were survival, maturity age (the time of the first egg appeared in the brood chamber of Daphnia), and reproduction. By the end of the test, all living Daphnia female were immediately fixed with Lugol solution [2], and body length was measured on the microscope (Olympus BX 51) coupled with a digital camera (DP71) to evaluate the growth of the animal. Besides, age-specific survival and clutch size were used to estimate the intrinsic rate of population increase, as a measure of fitness.

Sigmaplot version 12.0 was used for the data processing. Kruskal-Wallis test was applied for calculation on the statistically significant difference of the maturation, the fecundity and the intrinsic growth rate of D. magna. The Euler equation [28] was used to calculate the intrinsic rate of population increase:

Where:

x – age (in days)

l_x – the probability of surviving

m_x – the fecundity at age x.

As seen in Fig. 1, at the end of this experiment, the survival of D. magna in the control treatment reduced to 93% that was within the eligible range of values of chronic toxicity test according to Rice and Bridgewater (2012). Exposed to ampicillin at a concentration of 50 ppb, survival proportion of D. magna maintained at 100% during 18 days of exposure but became the same as the control treatment by the end of the experiment, day 21. However, survival of daphnids in 5 and 250 ppb exposures was gradually decreased to 67% and 47%, respectively, when the test terminated (Fig. 1). Our results were in line with the previous study of Dalla Bona et al. (2014) in which both enrofloxacin (at the concentration of 0.9 mg/L) and sulfaguanidine (at the concentration of 2.5 mg/L) reduced the survival of D. magna to less than 30%, whereas, ciprofloxacin (5 mg/L) and sulfaguanidine (3.2 mg/L) significantly reduced the survival of Daphnia curvirostris to less than 10% after 13 days of incubation. Besides, this result was in agreement with the investigation of Park and Choi (2008) revealing that the antibiotics of β-lactam class were toxic to Vibrio fischeri, D. magna, Moina macrocopa, and Oryzias latipes. Antibiotics could impact on bacteria-mediated digestion in D. magna leading to the limitation on nutrient breakdown and absorption in the animals [12], consequently energy cost. This may help to explain the mortality increase in ampicillin treatments in our study. Further investigations to confirm the negative influence of ampicillin to the useful bacterial communities in Daphnia digestive tract are suggested.

Figure 1.

The influence of ampicillin on the maturation of D. magna was showed in Fig. 2. Daphnia magna in 50 ppb treatment reached their maturation around the age of 4 days old, significantly earlier than the control and other ampicillin exposures (5 ppb and 250 ppb; p < 0.001), which took around 6 days. The stimulation on maturation in 50 ppb treatment could be an adaptation of daphnids under the unfavorable conditions [22]. It is notable that Ampicillin in several specific concentrations was not highly toxic to the aquatic organism and algae comparing to the other antibiotics [27]. Moreover, the comparable concentration of ampicillin could be impellent the maturation of D. magna which was showed in our study. However, the stimulation of maturation, which was presented by toxic substances, was able to lead to the serious issues at the next generation such as the death and the malformation of the neonates [21].

Figure 2.

In control, the clutch size of a mother D. magna was around 8 offsprings. However, the clutch size of D. magna was slightly increased in the 5 ppb and 50 ppb treatments. The average number of offspring per clutch in these treatments were approximately 9 neonates whereas the clutch size in 250 ppb treatment were decreased to 7 neonates (Table 1).

In addition, during three weeks of experiment, the total accumulative offspring in control was 602 offsprings. However, in comparison with the number of neonates in control, that in the 5 ppb and 250 ppb treatments decreased considerably to 75% and 32%, respectively. Regarding to brood number per female, there were a strong reduction from approximately 5 broods in control to around 3 and 2 broods in 5 ppb and 250 ppb treatment, respectively. Especially, because experimental organisms exposed 50 ppb ampicillin reached early maturity day, the total neonate and the brood number per female in this treatment were highest, 783 offsprings and around 6 broods. These results were in consistent with a previous study of Giordano et al. (2010) who discovered that ampicillin at high concentration reduced egg number of an invertebrate species, Folsomia candida.

At the end of the experiment, the average body length of female D. magna in the control was around 3.42 mm. In 5, 50, and 250 ppb ampicillin exposures, those decreased to 3.38, 3.37, and 2.98 mm, respectively (Fig. 3). The inhibition of growth or shorten body length in the 250 ppb exposure in this study was in line with a previous investigation in which ampicillin caused a significant decrease of Folsomia candida’s body length [11].

Figure 3.

As mentioned above, the ampicillin exposures strongly affected on survivor, maturity and reproduction of animals, consequently those effects on the intrinsic population rate. Daphnia in 5 ppb and 250 ppb treatments had a significantly lower intrinsic rate of population than that of the control experiments. The intrinsic population rate of Daphnia incontrol was 0.333 whereas those in 5 ppb and 250 ppb ampicillin treatments were comparable, 0.315 and 0.296, respectively (Fig. 4). On the other hand, the intrinsic growth rate of the daphnids in 50 ppb treatment was highest, reached 0.417 due to the earlier maturation of the daphnids. Ferrando et al. (1995) reported that population growth would be determined primarily by the frequency of the first few broods which was supported by our results (Table 1 and Fig. 4).

Figure 4.