Distribution and Elimination of Norfloxacin in Fenneropenaeus chinensis Larvae

SUN Ming1), 2), LI Jian2), *, ZHAO Fazhen2), LI Jitao2), and CHANG Zhiqiang2)



Distribution and Elimination of Norfloxacin inLarvae

SUN Ming, LI Jian, ZHAO Fazhen, LI Jitao, and CHANG Zhiqiang

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This study examined the distribution and elimination of Norfloxacin (NFLX) inovary and egg and newly hatched larvae. Mature parental shrimp were exposed to 4 or 10mgLNFLX for 2 or 5d. Ovary and eggs of the shrimp were sampled after spawning in order to detect NFLX residue using high-performance liquid chromatography (HPLC). Results showed that NFLX residue accumulated ineggs after the parental exposure, with the highest residue detected in ovary. To examine the fate of NFLX residue in larvae, we further determined the concentration of NFLX residue ineggs and larvae at 4 different developmental stages after 24-h exposure. From the newly metamorphosed larvae (0h post-metamorphosis, h.p.m), samples were taken at different time intervals to 72h.p.m. HPLC assay showed that the concentrations of NFLX residue in zoea exposed to 4 and 10mgLNFLX were the highest at 1.5h,., 0.332 and 0.454µgg, respectively. At the two NFLX exposure levels, the elimination time of half NFLX (half life) in nauplius was 45.36 and 49.85h, respectively, followed by that in zoea (31.68 and 33.13h), mysis larvae (42.24 and 47.28h) and postlarvae (24.48 and 30.96h). Both NFLX exposure levels had a germicidal effect. The distribution and elimination of NFLX residue in.tissue, eggs and larvae correlated well with the drug exposure level. The disappearance of NFLX residue coincided with the larval growth, and the half-life of NFLX decreased with the larval development.

larva; NFLX; HPLC; residue analysis

1 Introduction

is an economically important shrimp species that is commonly cultured in China (Wang., 2011) and distributed wildly along the coast stretching from Shandong Province of China to Korean Peninsula (Liu, 1959). Similar to other crustaceans,does not directly develop from the egg to adult-like juvenile but goes through a complex life style involving an egg phase, a larval phase, and a juvenile-adult phase. The primary larval phase, which consists of nauplius, zoea, mysis larvae and postlarvae, differs from juvenile-adult in specific traits such as morphological and behavioral features related to feeding and locomotion (Klaus, 2006).is of great importance to China’s mariculture industry and contributes to a total pond-production of 150000 t in 1991 (Wang., 2006).is ideally suited for mariculture because of its rapid growth, resistance to low temperature, superior nutritional property and easiness of adaptation to artificial culture conditions (Li., 2006). However, the incidence of epizootic diseases in shrimp cultivation has been increasing due to high density stocking and bacterial infections such as those byand(Zhang., 2004; Qin., 2006). Therefore, utilizing antimicrobials to prevent and control relevant problems has become an important part of shrimp farming.

Norfloxacin (NFLX), a ‘third-generation’ member of the fluorinated quinolone family, is a synthetic broad- spectrum antibacterial drug (Wang., 2008). It is widely used in human and veterinary medicine that targets both Gram-negative and Gram-positive bacteria through inhibition of DNA gyrase (Liu., 2011), and is extensively used to prevent and treat bacterial infections that may evoke egg mortality during the incubation period. In addition, NFLX has several toxic effects on mammalian cells (Sherri., 1998) and interferes with the hepatic metabolism. Previous studies have investigated NFLX in a number of fresh and seawater species such as freshwater shrimp (He., 2008), soft-shelled turtle (Chen., 1997), carp () (Zhang., 2000),(Fang., 2003),(Fang., 2004) and Japanese sea perch, and black sea bream (Wang., 2008). However, research of NFLX residue has mainly been constrained in adult shrimp (Sun., 2011), and few studies have addressed the distribution and elimination of NFLX from mature parental shrimp to the offspring. In the present study, we treated the parent mature shrimp with NFLX at two exposure levels before spawning, and then determined the concentration of NFLX residue in theovary, eggs and newly hatched larvae. To confirm the elimination of NFLX residue in larvae, we determined the disposition of NFLX residue ineggs and larvae at different developmental stages after 24-h exposure to NFLX and examined the disappearance of NFLX residue for as long as possible.

2 Materials and Methods

2.1 Chemicals

NFLX tablets were manufactured by Siming Medicine Company (Zhejiang, China). NFLX standard (purity 99%) was purchased from China Institute of Veterinary Drug Control (Beijing, China). Acetonitrile and methanol purchased from Merk were at high-performance liquid chromatography (HPLC)-grade; phosphoric acid and N- hexane purchased from Sinopharm Chemical Reagent Co., Ltd were at analytical grade.

2.2 NFLXExposure to Parent Shrimp and Shrimp Maintenance

Healthy, parent maturewere purchased from a local shrimp farm in Shandong Province, China. The parent shrimp were randomly divided into 36 tanks of 200 liters in volume, 2 individuals each tank. The salinity of sea water was 30, and the culture temperature was maintained at 18℃. Preliminary analysis of shrimp tissues proved a lack of NFLX in the shrimp prior to the experiment. Parent shrimp were exposed to NFLX at the levels of 4mgL(NFLX-4) or 10mgL(NFLX-10) for 2or 5d by water administration. At each NFLX level, 9 replicates were set. During NFLX exposure, the volume of seawater each tank was set to 160L. NFLX stock solution was added to the tank with a pipette to the desirable concentration. Then, the eggs were cleaned 3 times with freshwater, sterilized as soon as the parent shrimp ovulated, and further transferred into tanks containing sea water without NFLX for incubation.

2.3 NFLX Exposure to Shrimp Larvae and Larvae Maintenance

Healthy nauplius, zoea, mysis larvae and postlarvae ofwere obtained from a commercial fish farm in Shandong Province, China. The larvae at different developmental stages were incubated under the same condition as for parent shrimp, and then used for experimentation at the first development stage after metamorphosis. The larvae at each developmental stage were randomly divided into 6 tanks, 20g each. The salinity of sea water was 30, and the temperature was maintained at 21℃, 22℃, 24℃ or 24℃. Background NFLX residue was undetectable inlarvae prior to NFLX exposure. The larvae were exposed to 4 or 8mgLNFLX for 24h by water administration. Each NFLX exposure was done in three replicates. During NFLX exposure, the water volume each tank was set to 160L, and NFLX stock solution was added to the tanks with a pipette to obtain the desirable concentration,., 4 or 10mgL.

2.4 Parent Shrimp and Larvae Sampling

The ovary of parent shrimp and the ovulated eggs were sampled after spawning. The remaining eggs were incubated at 21℃ and sampled during the metamorphism to nauplius. One gram of larvae were randomly obtained each tank and then sampled at 0.5, 1, 1.5, 2, 3, 4, 6, 9, 12, 24, 36, 48 and 72h after NFLX exposure. The larvae were cleaned with sea water 3 times. All samples were stored at −20℃ prior to analysis. Control samples were assayed for NFLX and no NFLX was found in any shrimp tissues or larvae sampled before the initiation of the study.

2.5 NFLX Determination

NFLX extraction and analysis were carried out following the procedure of Sun. (2011). The ovary and larvae (1g each) were mixed with 2mL of acetonitrile, homogenized with a high-speed homogenizer at 16000rminfor 20s, and then centrifuged at 2655g for 10min. The supernatant was transferred to a clean test tube and the remaining tissue pellet was extracted with 2mL of acetonitrile. The extracts were combined and evaporated to dryness under nitrogen at 40℃, then re-suspended in 1mL of the HPLC mobile-phase solvent. Thereafter, 2mL of hexane was added and the mixture was vortexed to remove the fat. The aqueous phase was retained and filtered through a 0.22-μm filter unit. A 20-μL aliquot of the sample was injected into the Agilent 1100 HPLC system (USA). The column was operated at 30℃. The mobile phase was acetonitrile: 0.01molLphosphate buffer (16:84) at a flow-rate of 1.0mLmin.

2.6 Detection Limit and Recovery of NFLX from Ovary and Larvae

The recovery of NFLX from the ovary and larvae was determined using control samples spiked with 0.1, 0.5, 1, or 5μgmLNFLX and extracted as previously described. The sample homogenate or solution was allowed to interact with NFLX at 4℃ for at least 2h before the extraction. The assay method was validated for the reaction system and the method precision was evaluated by analyzing one sample five times on the same day and five individually prepared samples over five separate runs on different days. Results showed that the assay method was robust for the system, with high precision as reflected by the low relative standard deviation (SD). The intra- and inter-day precision of the analytical method were (2.37± 1.21)% and (3.46±1.52)%, respectively.

2.7 Statistical Analysis and Estimation of NFLX Half-Life

Data are presented as the mean ± SD (=5). The terminal phase of the elimination-time curve was analyzed by a linear regression analysis on the log-transformed concentration-values versus time. Elimination curve equations and the elimination rate constant (ß) were obtained from a computer fitting program. The half-life () of NFLX in shrimp tissue was calculated as follows:= 0.693/ß (Baggot, 1977).

3 Results

3.1 NFLX Analysis and Recovery

In this study, a sensitive and specific HPLC method was used to quantify NFLX residue inovary and larvae. The response of HPLC-UV detector was linearly correlated to the concentration of NFLX standard up to 50μgmL. The detection limit of NFLX in theovary and larvaewith HPCL method was 0.01 µgg. Percentage recovery of NFLX from the ovary and larvae at different developmental stages were (89.54±3.03)% (ovary); (79.62±2.82)% (nauplius); (73.29±2.17)% (zoea); (72.65±2.58)% (mysis larvae); and (71.84±2.73)% (postlarvae).

3.2 NFLX Residue in Parent MatureOvary and Nauplius

In the newly hatched eggs and developing larvae, NFLX residue was detected at high levels (Table 1). The mean concentration of NFLX residue in ovary clearly decreased with the reduction of NFLX dosage and expo-sure time. The corresponding level of NFLX in eggs and larvae varied in a similar trend.

Table 1 The concentration of NFLX residue in ovary, newly hatched eggs and nauplius after NFLX exposure

The fate of NFLX residue in shrimp eggs and nauplius was similar to that in the ovary, and the level of NFLX residue was lower in nauplius than in eggs with the development of the larvae and the metabolism of the organism. The detection of NFLX residue in newly hatched eggs and the developing larvae indicated that NFLX can penetrate through the eggshell into the embryo of.

3.3 NFLX Residue inat Different Developmental Stages

After NFLX administration, the concentration of NFLXresidue in nauplius peaked at 1.5h in the NFLX-4 treatment (0.122µgg) and 0.5h in the NFLX-10 treatment (0.409µgg). The concentration of NFLX residue in zoea peaked at 1.5h in both the NFLX-4 (0.332µgg) and NFLX-10 treatments (0.454µgg) (Fig.1). For mysis larvae, the concentration of NFLX residue peaked at 1.5h in the NFLX-10 treatment (0.331µgg), nearly 10-fold higher than that at 0.5h in the NFLX-4 treatment (0.036µgg). The NFLX absorption peak of postlarvae was observed at 0.5h in both the NFLX-4 (0.209µgg) and NFLX-10 treatments (0.306µgg). The NFLX residue in larvae was positively correlated with exposure concentration. The residue concentration detected in all developmental stages of larvae was much lower than the exposure concentration. NFLX residue in zoea was higher than other developmental stages of larvae at both 4 and 10mgL, which may be related to the physical structure and drug metabolizing ability.

Fig.1 The concentration of NFLX residue inlarvae at different developmental stages after exposure. a, nauplius; b, zoea; c, mysis larvae; d, postlarvae.

3.4 Elimination of NFLX inLarvae

The half-life of NFLX and the corresponding correlation coefficient following water administration of NFLX at different dosages are shown in Table 2. The decrease in NFLX concentration is represented by the elimination equation. The half-life of NFLX in larvae decreased in the order nauplius>mysis larvae>zoea>post-larvae at both 4 and 10mgL. There was no influence of exposure concentration on half-life of NFLX.

Table 2 Elimination equations and parameters of NFLX residue in administration of NFLXFenneropenaeus chinensis larvae following water

4 Discussion

4.1 The Pharmacodynamics of NFLX

In the spawning period, vibrio diseases are mainly caused by pathogenicsuch as, and. Shrimp larvae bacteremia commonly takes place due to the mixed infection of thesespecies that mainly occur in hemolymph (Yu., 2000). NFLX is a common fluorinated quinolone drug and exerts a rapid sterilization effect by acting on DNA gyrase and disturbing DNA synthesis of bacteria. NFLX has been used to treat bacterial diseases such as bleed, lousy gills, enteritis, ascites, and sepsis infish, prawns, crabs and turtle caused by,,or(Yang., 2004). Research has examined the anti-bacteria effects of NFLX on specific bacterial species. The inhibiting and bactericidal effects of NFLX were found varying and species-dependent. For instance,was found highly sensitive to NFLX at 8–16mgL(Wang., 2008). The minimum bactericidal concentration (MBC) of NFLX againstwas estimated to be 0.16µgmL, and the pesticide effect of NFLX was shown increasing with the drug concentration (Chen., 2010). In the present study, when administered in different dosages for 2 or 4d, NFLX residue showed a sterilization effect onto different degrees, with the concentrations in ovary higher than MBC. The concentrations of NFLX residue in eggs were higher than MBC except for the eggs from parent shrimp exposed to of 4mgLNFLX for 2d. The concentrations of NFLX in nauplius were lower than MBC, indicating the lack of a sterilization effect. Together these findings indicated that the drug should be administered at proper concentrations for preventing pathogenic bacteria from giving rise to resistance to sub-lethal concentrations.

4.2 Transportation of NFLX from Mature Shrimp to Offspring

This study presented the first evidence on the distribution and transportation of NFLX from mature parent shrimp to offspring. Data analysis indicated that NFLX transferred from parent shrimp to the offspring and existed in both eggs and nauplius. The amount of NFLX residue increased with the drug dosage and the exposure time. The reproductive mode ofis oviparity. The detection of NFLX residue in eggs and newly hatched larvae indicated that NFLX in the ovary is capable of penetrating through eggshell into the oocyte. As a carrier of hydrophobic fluorine atoms, NFLX can penetrate through plasma membrane and spread quickly, thus enhancing the lipophilic and tissue penetration (Yi., 2010). Drugs with molecular weight between 250 and 500Da are easily diffused through cell membrane. Thus, it is easy for NFLX (MW 319.34Da) diffusing through cell membrane.

Despite a number of previous studies on the biological transport of NFLX in human cellular, few studies have addressed the distribution of NFLX in shellfish. Biological transport of NFLX has been confirmed in human periodontal ligament fibroblasts (Liu., 2008). Koga (1987) determined the transport of NFLX in neutrophils and found that the concentration ratio of intracellular to extracellular NFLX was 2.2. Tulkens (1991) pointed out that almost all of the fluorinated quinolone antibiotics can be transported to macrophage.

Distribution of other drugs in mammalian has been studied in detail. For mammalian, placenta plays the most important role in the fate of drugs. Vasculo-syncytical membrane, the transporter of drug in placenta, is a thin film that consists of syncytiotrophoblast with basal membrane, fluffy interstitial and capillary endothelial cells. The transport patterns of drugs in placenta mainly contain simple diffusion, active transport, and pinocytosis. Most drugs can access the embryo through placental barrier. In pregnancy rats administered with 100mgkgcefotaxime, residual cefotaxime was detected at relatively low levels in fetal rats while the plasma cefotaxime concentration was 11µgmLat 5min and peaked at 20min (Zhang., 1999). In lamb injected with a single dose of salvia miltiorrhiza (800, 400, or 200mg), protocatechualdehyde, an effective ingredient of salvia miltiorrhiza, was detected in the plasma of fetal lamb at 0.083, 0.5, 1.5h. The drug dosage was found relatively positively correlated with the plasma protocatechualdehyde concentration, indicating that protocatechualdehyde was transport into fetal lamb through placental barrier (Shao., 2009). In pregnant rats intraperitoneally injected with VO, vanadium was found accessing embryo/fetal rats through placental in both the embryonic period and the fetus period, despite that fetal rats had a certain barrier function with vanadium (Zhang., 1991). Organic and inorganic mercury can both access the fetus through placental pathway, and the former is easier to transfer than the latter (Mansour., 1973). There is a p-glycoprotein mainly expressed in tissues with a barrier structure, such as the blood-brain barrier, liver, kidney, and placenta. P-glycoprotein has a pumping function and mediates the transport of drugs out of cells. In associated physical processes, p-glycoprotein mainly participates in the absorption, distribution, and excretion of drugs. NFLX has been found significantly increasing the p-glycoprotein expression in rats (WU., 2004). Although NFLX can transport from parent shrimp through the ovary, it is unclear whether p-glycolprotein exists inovary. Further study is recommended to confirm whether there is a barrier in.

4.3 Accumulation and elimination of NFLX inLarvae

Determination of NFLX residue intissues, eggs and larvae showed that more than 70% of NFLX administered to the larvae was recovered by acetonitrile and well separated with miscellaneous peaks. NFLX effectively penetrated into the larvae, thus was in favor of killing pathogenic bacteria.

After NFLX exposure, the residual drug concentrations in zoea were the highest, 0.332µggµggh in the NFLX-4 treatment and 49.85h in the NFLX-10 treatment; whereas those of NFLX in post- larvae were the shortest, 24.48h in the NFLX-4 treatment and 30.96h in the NFLX-10 treatment. These observations could be explained by the gradually developed structure with the metamorphosis and development of the larvae. Considering the incomplete mouthpart and inchoate alimentary canal, the naupius was supplied with yolk due to a lack of feeding. Thus, the drugs were absorbed by osmosis. Jia. (2000) investigated the absorption and utilization of dissolved organic matter (DOM) innauplius yet further research is needed to investigate which organ is used to absorb DOM. In the present study, the mouthpart and alimentary canal were formed in the period of zoea, and the drug was absorbed by both osmosis and filterfeeding. This led the concentration of NFLX residues to be higher in zoea than in nauplius. With the metamorphosis and development of the larvae, the structure of mysis and post-larvae was more developed, especially the blood circulation system. There is only an artery sent out from heart in zoea, while several arteries exist in mysis. Blood is supplied to different tissues by these arteries, contributing to the gradual development of larvae and the differentiation of functions (Ma., 2000). The concentrations of NFLX residue in mysis and post-larvae were lower than in zoea due to the increase in drug metabolising ability. This explains why the half-life of NFLX was the longest in nauplius and the shortest in post-larvae. The half-life is a significant fixed constant for evaluating the elimination rate of drugs in an animal. It is only influenced by the nature of the drug, but not the drug dosage (Ding., 2000). Research indicates that the half-life of drugs is species-dependent in animals,., it is shorter in homeotherm and longer in poikilotherm (Yang., 2005). The plasma half-life of NFLX in Hubei white and AA dorking was 3.34 and 3.49h, respectively (Cao., 1997). The results in the present study indicated that the half-life of NFLX in larvae at different developmental stages is longer than 20h, and that the NFLX elimination rate is slower in poikilotherm than in homeotherm. The metabolic differences of NFLX in different animals may be related to their evolution level. There are more developed drug excretion systems and organs as well as perfect drug elimination mechanisms. Drugs that enter an organism can be actively eliminated by the kidney, thus accelerating the elimination rate of NFLX. In lower aquatic creatures, drugs are passively eliminated by unperfected organisms (Nouws., 1988), resulting in the relative low elimination rate.

5 Conclusions

The present study examined the fate of NFLX residue intissue, eggs, and larvae post-NFLX exposure. Results showed that NFLX could transport from mature parent shrimp to offspring. The accumulation and elimination of NFLX residue in shrimp ovary, eggs, and larvae correlated well with the level of drug exposure. Further study is needed to evaluate the influence of NFLX residue on the hatching rate ofeggs. The disappearance of NFLX residue co-occurred with the growth of the larvae, and the half-life of NFLX decreased with the development of larvae.

Acknowledgements

This study was supported by the Earmarked Fund for Modern Agro-industry Technology Research System, China (No. CARS-47), and the Special Fund for Agro-scientific Research in the Public Interest of China (No. 201103034).

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(Edited by Qiu Yantao)

10.1007/s11802-013-2179-8

ISSN 1672-5182, 2013 12(3): 397-402

. E-mail: lijian@ysfri.ac.cn

(October 23, 2012; revised November 27, 2012; accepted June 25, 2013)

© Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2013