Chronic exposure to organic oxygen-demanding pollutants at an environmentally realistic concentration affects sperm motility in zebrafish
Graphical abstract
Introduction
Organic oxygen-demanding pollutants (ODPs), also known as oxygen-consuming organic substances, are usually classified into three major categories, namely, carbohydrates, proteins, and fats. ODP, such as sugars, proteins, oils, amino acids, fatty acids, and esters, are commonly found in domestic sewage and various industrial wastewaters (Osibanjo, 2007; Ding and Rong, 2016; Dadi and Stellmacher, 2017). ODP is one of the most common types of water pollutants. The discharge of sewage from the slaughterhouse into the river leads to a decline in species richness and diversity and reduces the ability of fish to resist oxidative stress (Olaniran and Sogbanmu, 2019).
With rapid economic development, the ODP pollution in rivers has become extensive. Various factories, such as slaughterhouses, and industries, such as potato processing, olive oil, cheese production, beer production, and abattoir, are involved in producing large quantities of ODPs worldwide (Ziara, 2016; Olaniran and Sogbanmu, 2019). The most serious ODP pollution is found in the Ziya River basin, one of the seven major river basins in China (Wang, 2012; Rong and Shan, 2016a,b). COD is one of the commonly used parameters of organic pollution and reflects the extent of ODP contamination in water. The range of chemical oxygen demand (COD) fluctuates between 27.50 and 410.00 mg/L (Ding and Rong, 2016). The COD concentration range of the drinking water source protection area in Guiyang City, China was 0.01–160.00 mg/L in 2010 and dramatically increased to 12.00–140.00 mg/L in 2011 with an average of 35.42 mg/L (Wang and Yin, 2019). This value is higher than the upper limit for COD concentration in the third and fifth types of surface water. As such, the surface runoff in the basin is seriously polluted by ODP.
ODP has received attention because it may affect the richness of fish or other aquatic organisms through the consumption of dissolved oxygen in the water. Dyer, S. D. et al., considered that the decrease in COD caused by untreated wastewater may have an adverse impact on the Balatuin River in the Philippines, and investigated the diversity of aquatic communities, and found that low dissolved oxygen is one of the reasons for poor colonization from upstream and downstream reaches of the river (Dyer and Peng, 2003). However, the effect of ODP on the reproductive ability of aquatic organisms has not been reported. We used the zebrafish model to explore the effects of ODP exposure on the general physiological status and reproductive health of male zebrafish. We simulated the composition of ODP (carbohydrate, protein, and fat) by adding egg yolk powder (containing a small amount of maltose, a large amount of protein, and fat) to the zebrafish culture system. The natural purification of ODP-contaminated water bodies was simulated by adjusting the flow rate of circulating water in the zebrafish culture system. The physiological status and reproductive health of zebrafish were evaluated by measuring body length, body weight, blood sugar, blood cholesterol, liver Oil Red O, sperm motility, fertilization rate, and offspring hatching rate. Sperm motility was measured by the Computer Assisted Sperm Analysis system. In addition, we examined the transcriptional changes of immune-related genes, including the classical cytokines tnfα, il-1β, il-8 and il-4 in fish male gonads. The present study focused on determining the effect of ODP on physiological status and reproductive health in zebrafish. We speculate that ODP may adversely affect the reproductive health of aquatic organisms by promoting inflammation.
Section snippets
Chemicals and materials
Egg yolk powder (98 % purity) was purchased from Solarbio (Cat#E8200, China), stored at room temperature, and dissolved in fish water. Each adult fish was exposed to ODP with a final concentration of 30 mg/L COD. Fragments and cover slips for zebrafish sperm motility testing were purchased from Hamilton Thorne (Lot # 559599, USA). Oil Red O was purchased from Sigma (Lot # SLBT6544, USA). Reconstituted fish water was prepared as follows. Deionized water was added with ions (34.0 g/L NaCl,
Detection of COD and blood glucose and cholesterol test results
COD is one of the commonly used indicators of ODP. During the simulated ODP pollution, the original fish tank showed evident turbidity. The anoxic zebrafish behavior of swimming to the upper layer was not observed, and water turbidity gradually decreased. After 2 h, the breeding environment of the ODP group did not show a significant difference compared with that of the control group. The mode diagram of the simulated ODP environment is shown in Fig. 1A. Fig. 1B shows the COD content of the
Discussion
Zebrafish is a commonly used experimental animal for evaluating water pollution (Parng, 2005; Ajagbe, 2012), and is also a well-recognized vertebrate model for research development and ecotoxicology(Segner, 2009). Therefore, we used zebrafish to evaluate the biological toxicity of ODP in the present work. ODP contamination is known to induce toxic effects in aquatic biota, including by reducing populations of aquatic animals (Dyer and Peng, 2003; Olaniran and Sogbanmu, 2019), but its
Conclusions
Although the general physiological condition of the zebrafish exposed to ODP (liver Oil Red O, blood sugar, blood cholesterol, subcutaneous fat, body weight, and body length) and the fertilization rates in zebrafish embryos did not show a statistical difference, the level of serum testosterone protein was decreased, the embryo incubation time was delayed, and the expression of pro-inflammatory factors in male zebrafish gonads was dramatically increased. In summary, while the up-regulation of
Credit author statement
YS and CL designed research studies, conducted experiments, analyzed data, and drafted the manuscript. LH conducted Zebrafish model establishment. KZ conducted sample collection and storage. HZ provided intellectual input into planning of experiments and ZM contributed to the writing of the manuscript.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
This study was supported by The Open Research Fund of NHC Key Laboratory of Birth Defect for Research and Prevention (Hunan Provincial Maternal and Child Health Care Hospital) (KF2018001), The National Key Research and Development Program of China (2018YFC1004300) and Wuhan Youth Science and Technology Morning Project (2017050304010291).
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