Chemosphere 139 (2015) 229–234
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Biomarker analysis of combined oxytetracycline and zinc pollution in earthworms (Eisenia fetida) Minling Gao ⇑, Yun Qi, Wenhua Song, Qian Zhou Department of Environmental and Chemical Engineering, Tianjin Polytechnic University, No. 399 Binshui Western Road, Xiqing District, Tianjin 300387, China
h i g h l i g h t s exposure were proofed compared to Zn2+ alone. 2+ A mechanism involving complexation of Zn and OTC at alkaline pH is proposed. Lysosomal membrane stability and coelomocyte apoptosis are sensitive biomarkers. Antagonistic effects of OTC and Zn
a r t i c l e
2+
i n f o
Article history: Received 17 March 2015 Received in revised form 11 June 2015 Accepted 12 June 2015
Keywords: Coelomocyte apoptosis Earthworm Interactive effect Lysosomal membrane stability Antibiotic Heavy metal
a b s t r a c t To determine the interactive action of antibiotics and heavy metals, this study assessed pollutant-induced responses of cellular biomarkers in earthworms (Eisenia fetida) exposed to zinc (Zn2+) and oxytetracycline (OTC) in soil. Lysosomal membranes were damaged and coelomocyte apoptosis occurred with exposure to the individual and combined pollutants. Compared with Zn2+ alone, lysosomal membrane stability and coelomocyte apoptosis decreased in the Zn2+–OTC combined treatment, possibly as a result of complexation of Zn2+ and OTC at alkaline pH. Such complexation could reduce the toxicity of the pollutants. Lysosomal membrane stability and coelomocyte apoptosis are sensitive biomarkers and could be economical and rapid tools for the monitoring and assessment of a variety of pollutants. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The antibiotic oxytetracycline (OTC) is used extensively worldwide in veterinary drugs and feed additives, and is beginning to pollute soils. Most veterinary antibiotics cannot be completely absorbed by animals and are discharged through feces and urine as the parent compound or as metabolites (Halling-Søensen, 2001; Liao et al., 2001; Kong and Zhu, 2007). The impact of OTC on the environment is increasing with the development of intensive livestock and poultry breeding programs and the wide use of manure fertilizer. The average OTC content is as high as 9.1 mg kg 1 (1.1–135 mg kg 1) in pig manure and 6.0 mg kg 1 (2.9–23 mg kg 1) in chicken manure (Zhang et al., 2005). OTC concentrations averaged 5.2 mg kg 1 in surface soils (0–20 cm) from agricultural fields treated with animal manure in northern Zhejiang Province, China (Zhang et al., 2008). Hamscher et al. ⇑ Corresponding author. E-mail addresses: [email protected] (M. Gao), [email protected] (Y. Qi), [email protected] (W. Song), [email protected] (Q. Zhou). http://dx.doi.org/10.1016/j.chemosphere.2015.06.059 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
(2000) showed that contents of oxy- and chlortetracycline in surface soils (0–40 cm) were as high as 32 and 26 mg kg 1, respectively, after application of manure fertilizer. Tetracyclines, including OTC and other veterinary antibiotics, may lead to antibiotic resistance in microorganisms, which is potentially hazardous to non-target organisms in terrestrial environments. Various effects of OTC have been observed, including the inhibition of bacteria, actinomycetes, and total microorganism populations (Wang and Zhang, 2009); decreased urease, sucrose phosphatase, and hydrogen peroxidase activity (Yao et al., 2010); reduced root and shoot elongation in wheat and Chinese cabbage (Jin et al., 2009); and genotoxicity to earthworms (Dong et al., 2012). Zinc (Zn) is a trace element that is required for biological growth. The content of Zn in soil generally ranges from 10 to 300 mg kg 1 (Li et al., 2006), but Zn concentrations exceeding 13,500 mg kg 1 have been reported for polluted soil in China (Li et al., 2006). Some plants can absorb and accumulate Zn, but excess zinc uptake can inhibit plant growth and development (Song et al.,
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2003; Lin et al., 2007). Zinc can also affect soil respiration (Vanhala and Ahtiainen, 2006), enzyme activity (Yang et al., 2006; Lin et al., 2007), and bacterial diversity and population size (Moffett et al., 2003; Lu et al., 2010), and can have negative effects on soil and human health (Lock and Janssen, 2001; Jia et al., 2005). Antibiotics and heavy metals are often detected together in soils and organic fertilizers (Zhang et al., 2005). Combined antibiotic and heavy metal pollution has received increasing global attention. For example, metal- and antibiotic-resistant bacteria were found in Antarctic marine waters, and this was attributed to long-term exposure to low concentrations of these pollutants (De Souza et al., 2006). Ecotoxic effects of antibiotics in soil have been reported (Thiele, 2005; Bao, 2008; Hammesfahr et al., 2008; Liu et al., 2009). However, little information is available on the potential biochemical and genetic effects of veterinary antibiotics and heavy metals in soils, although a few studies have indicated that the combination of antibiotics and metals can be harmful to environmental bacteria and earthworms (Kong et al., 2006; Gao et al., 2013, 2014). More information on the effects of these pollutants is needed for improved soil risk assessment. Biomarkers in terrestrial invertebrates can be used to estimate the exposure to and effects of pollutants. Earthworms are sensitive indicators of soil quality and are widely used in terrestrial ecotoxicology (ISO, 1998; Lin et al., 2007). Many biomarkers have been utilized to profile the toxicological effects of pollutants, including lethality, LD50, reproductive rate, enzymatic activity, lysosomal membrane stability, cell apoptosis, and DNA damage (Sforzini et al., 2012; Wu et al., 2012). However, mortality, growth rate, and reproduction are commonly considered inappropriate for evaluating chemicals of lower toxicity and concentration. Qu et al. (2005) reported that OTC does not induce earthworm mortality at a concentration of OTC 500 mg/L. However, using the comet assay, these authors showed that OTC induced significant DNA damage in earthworm coelomocytes (P < 0.01). At the subcellular level, the stability of lysosomal membranes in coelomocytes can be used as a biomarker for the toxicological effects of contamination. One method that can be used to determine lysosomal membrane stability is the neutral-red retention assay. Neutral red retention time (NRRT) is a sensitive marker in earthworms that meets soil pollution risk assessment requirements (Svendsen et al., 2004; Jia et al., 2005). Flow cytometry accurately detects cell survival and apoptosis rates and reflects damage to cell membranes. This technique has been widely used to evaluate the toxicological effects of pollutants. Gao et al. (2014) reported a synergistic effect of 5 mg kg 1 OTC + 50 mg kg 1 lead (Pb) on earthworm lysosomes and they found antagonistic effects at higher concentrations (10–20 mg kg 1 OTC + 50 mg kg 1 Pb). In addition, coelomocyte apoptosis decreased significantly with combined OTC and Pb treatment compared with OTC alone, indicating an antagonistic reaction. In general, the modes of interactive effects between chemicals include antagonism, addition, and synergism. However, there is little information available on the interactive action of antibiotics and heavy metals. Knowledge of such interactions is important for assessing the toxicity, migration and transformation of pollutants, particularly compound pollution. Therefore, the objectives of this study were to (1) determine the interactive action of OTC and Zn2+ on earthworms, and (2) investigate the use of cellular biomarkers in earthworms (Eisenia fetida) exposed to zinc alone and in combination with OTC. We accordingly investigated lysosomal membrane stability and coelomocyte apoptosis for their potential use as sensitive, simple, and rapid biomarkers for soil monitoring and assessment.
2. Materials and methods 2.1. Soils and chemicals Surface soils (0–30 cm) were sampled from suburban farmland in Tianjin, China. The samples were air-dried, crushed to pass through a 10-mm-mesh screen, and stored in containers at 4 °C. The background contents of OTC and Zn were 0.0 and 8.3 mg kg 1, respectively. The soil had a medium loam texture with a density of 1.3 g cm 3; a pH of 8.2; an organic matter content of 1.6%; a water-holding capacity of 42%; a composition of 21% sand, 50% silt, and 29% clay; and a cation exchange capacity of 11 cmol kg 1. Oxytetracycline hydrochloride (95%–105% purity), EDTA, and dimethyl sulfoxide (DMSO, >99% purity) were obtained from Amresco (Ohio, USA). Guaiacol glyceryl ether (>98% purity) was purchased from Sigma (Beijing, China). Zinc nitrate (>99% purity) was obtained from Bodi Chemical Co. Ltd. (Tianjin, China). Lumbricus balanced salt solution (LBSS) consisted of 71.5 mM NaCl, 4.8 mM KCl, 3.8 mM CaCl2, 1.1 mM MgSO47H2O, 0.4 mM KH2PO4, 0.3 mM Na2HPO47H2O, and 4.2 mM NaHCO3. 2.2. Earthworms Earthworms (E. fetida) were purchased from Tianjin Chenggong Earthworm Farm Company, China. The worms were preincubated in clean soil for 1 week under controlled conditions (22 ± 2 °C, 12 h light:12 h dark, 75% humidity) before the experiment. Adult earthworms with a developed clitellum were collected for testing; the individual wet weight of adults ranged from 300 to 500 mg. 2.3. Experimental design Zinc nitrate at a concentration of 0, 50, 100, or 200 mg kg 1 was added to beakers containing 500 g soil. Oxytetracycline hydrochloride at a concentration of 5, 10, or 20 mg kg 1 was added to beakers containing 500 g soil and 50 mg kg 1 Zn. These dosages were chosen based on reported concentrations of OTC and Zn in soil. Moisture content was adjusted to 60% of the water-holding capacity. All tests were performed in triplicate. The soil was allowed to stabilize for 24 h prior to earthworm introduction. Ten worms were placed in each beaker and the beakers were enclosed with plastic film perforated with small ventilation holes. The test was conducted under a photoperiod of 12 h light:12 h dark at 20 ± 2 °C for 28 days. Humidity was maintained at 75% ± 5% by an artificial climate chamber. Worms were fed rewetted oven-dried cow manure (0.5 g per worm) every 7 days thereafter over the 28-day period. In order to maintain the original moisture percentage, distilled water was added gravimetrically to the replicates every week. Two earthworms were collected from each beaker without replacement at 7, 14, and 28 days. Although there was no earthworm death during the experiment, there was a significant difference in body weight loss among the different groups of earthworms. 2.4. Harvesting of coelomocytes Earthworms were allowed to empty their guts for 18 h in Petri dishes on filter paper moistened with distilled water. Each worm was rinsed with distilled water and saline (6.5 mg mL 1 NaCl, 4 °C) before being placed into a 1.5-mL Eppendorf tube containing 1 mL cold extrusion medium for 2 min. The extrusion solution contained 5% ethanol, 95% saline, 2.5 mg mL 1 EDTA, and 10 mg mL 1 guaiacol glyceryl ether and was adjusted to pH 7.3 with 1 M NaOH. Earthworms were removed from the tubes and the cell suspension
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was set aside for 10 min. Part of the cell-containing supernatant (0.5 mL) was transferred to another tube, and combined with LBSS. After centrifugation (100g and 4 °C for 3 min), the precipitate containing cells was collected and suspended in distilled water, adjusted to pH 7.3 with NaOH (Goven et al., 1993). Recovered cells were centrifuged at 100g and 4 °C for 3 min and then re-suspended in LBSS. Cell viability (trypan blue exclusion method) was assessed immediately after extrusion and was approximately 90% in all cases.
2.5. Neutral-red retention time Lysosomal membrane stability was assessed according to Svendsen et al. (1996). Briefly, 20-lL coelomic fluid samples were placed on a microscope slide and the cells were allowed to adhere to the glass surface for 30 s. The slides were then stained with 20 lL neutral red working solution (80 mg mL 1), which contained 2.5 mL LBSS and 10 lL neutral red stock solution. The neutral red stock solution was freshly prepared by dissolving 20 mg neutral red powder in 1 mL DMSO. The working solution was renewed every hour to avoid crystallization. Cover slides were placed and the slides were observed every 2 min under a microscope at 400; the slides were maintained in the artificial climate chamber between observations. Observations were stopped when the ratio of cells with fully stained cytosol was greater than 50% of the total number of cells counted. This time is referred to as the neutral red retention time.
7d 14d 28d
NRRT/min
50 40 30
** **
20
**
10
** ** **
** ** **
0 ck
50
100
Coelomocyte samples were washed twice with cold phosphate-buffered saline and then were suspended repeatedly with 200 lL 1 binding buffer. Subsequently, 100-lL aliquots of cell suspension were transferred to 1.5-mL Eppendorf tubes containing 10 lL Annexin V-PE. After mixing by inversion, the tubes were placed in an ice bath for 20–30 min in the dark. Subsequently, 380 lL 1 binding buffer and 10 lL 7-AAD dye were added to the cell suspensions and mixed gently. Finally, the cell samples were analyzed using a FACS Calibur flow cytometer (BD Biosciences, USA). 2.7. Statistical analysis To evaluate the effect of chemical exposure on earthworms, the NRRT inhibition rate was calculated as follows: NRRT inhibition rate = (NRRTcontrol group NRRTexposure group)/NRRTcontrol group. All data are presented as means ± standard deviation (SD). Significant differences between treatments were analyzed by one-way analysis of variance (ANOVA) using SPSS version 16.0 (SPSS, Inc., Chicago, IL). 3. Results 3.1. Combined effects of OTC and Zn on lysosomal membrane stability Compared to the control, the NRRT of coelomocyte lysosomal membranes decreased significantly (P < 0.01) after 7 days of exposure to Zn, and this affect increased with increasing Zn concentration (P < 0.05) (Fig. 1). Zinc exposure had a similar effect on lysosomal membranes after 14 and 28 days, and the effect was concentration dependent and highly significant (P < 0.05) at 200 mg kg 1. The NRRT inhibition rate at days 14 and 28 was 72% ± 5.6% and 80% ± 3.1%, respectively (Table 1). In contrast, NRRT decreased with increasing incubation time. The lowest inhibition rates occurred on day 14 and were 52% ± 9.4% (Zn = 50 mg kg 1), 68% ± 4.0% (Zn = 100 mg kg 1), and 72% ± 5.2% (Zn = 200 mg kg 1) (Table 1). Membrane stability decreased significantly in worms exposed to the combination of OTC and Zn (P < 0.05). Compared to Zn alone, exposure to combined OTC and Zn enhanced the NRRT inhibition rate with increasing incubation time (P < 0.05) but generally decreased the NRRT inhibition rate as OTC concentration increased (Fig. 2). The NRRT inhibition rate increased by 19%, 43%, and 78% at the dose of 5 mg kg 1 OTC + 50 mg kg 1 Zn, 10 mg kg 1 OTC + 50 mg kg 1 Zn and 20 mg kg 1 OTC + 50 mg kg 1 Zn at day 7 compared to the inhibition rate by Zn alone. Moreover, the
70 60
2.6. Flow cytometry
200
Zn (mg/kg) Fig. 1. Neutral red retention time of the earthworm exposed to single Zn.
Table 1 Combined effect of zinc (Zn) and oxytetracycline (OTC) on neutral red retention time (NRRT) inhibition rate. Time (days) 7
14
28
OTC concentration (mg kg 1)
NRRT inhibition rate (%)
Zn (mg kg
CK 5 10 20
58 ± 5.0 35 ± 4.1 54 ± 1.4 84 ± 1.4
CK 5 10 20 CK 5 10 20
NRRT inhibition rate (%)
OTC + Zn concentration (mg kg 1)
NRRT inhibition rate (%)
Combined effect
50 100 200
65 ± 6.2 69 ± 1.5 78 ± 3.1
5 + 50 10 + 50 20 + 50
81 ± 7.9 77 ± 11 71 ± 3.4
Antagonism Antagonism Antagonism
36 ± 4.5 53 ± 4.6 60 ± 5.5 75 ± 5.5
50 100 200
52 ± 9.4 68 ± 4.0 72 ± 5.6
5 + 50 10 + 50 20 + 50
80 ± 4.3 68 ± 11 43 ± 8.6
Antagonism Antagonism Antagonism
19 ± 4.0 42 ± 9.6 48 ± 5.8 67 ± 4.4
50 100 200
64 ± 9.1 72 ± 5.2 80 ± 3.1
5 + 50 10 + 50 20 + 50
72 ± 14 77 ± 14 61 ± 3.1
Antagonism Antagonism Antagonism
1
)
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4. Discussion
70
7d 14d 28d
60
NRRT/min
50 40 30
** 20
** 10
** ** ** * *
** **
**
0 ck
5+50
10+50
20+50
OTC+Zn (mg/kg) Fig. 2. Neutral red retention time of the earthworm exposed to combined pollution of OTC and Zn.
NRRT inhibition was strongest after 14 days of exposure to OTC + Zn; it was increased by 25%, 43%, and 83% at the dose of 5 mg kg 1 OTC + 50 mg kg 1 Zn, 10 mg kg 1 OTC + 50 mg kg 1 Zn and 20 mg kg 1 OTC + 50 mg kg 1 Zn compared to that under the corresponding Zn-only treatments (Table 1). 3.2. Combined effects of OTC and Zn on coelomocyte apoptosis Coelomocyte apoptosis in earthworms increased significantly in the Zn-only treatments (P < 0.05) (Table 2). Early and late apoptosis increased with increasing Zn concentration after 7 days of exposure and reached maximums of 31% and 24%, respectively, at 200 mg kg 1 Zn (Table 2). Late apoptosis generally increased (P < 0.01) with increasing Zn concentration after 14 days of exposure and reached 58% at 200 mg kg 1 Zn. After 28 days of exposure, early and late apoptosis rates were 19% and 38%, respectively (Table 2). Compared to Zn alone, coelomocyte apoptosis was significantly higher in earthworms exposed to OTC + Zn (P < 0.05) and the effects were dependent on OTC concentration (Table 4). However, coelomocyte apoptosis rates under the combined pollutants were lower than the sums of Zn-only and OTC-only (Table 3).
Although earthworm biomarker responses to heavy metals (Bundy et al., 2007; Annamaria Rocco et al., 2011; Calisi et al., 2013) and organic chemicals (Calisi et al., 2011) have previously been profiled, few studies have examined the effects of organic pollutants and heavy metals in combination. Earthworms provide an early warning of adverse environmental conditions (Calisi et al., 2013). The effects of pollutants at the cellular or subcellular level should occur before, or at lower concentrations than, effects at the population, community, or ecosystem scale (Svendsen et al., 1996). Thus, cellular dynamics are increasingly examined when evaluating the effects of xenobiotic pollutants on soil organisms. In the present study, lysosomal membrane stability and coelomocyte apoptosis were examined as cell biomarkers for the ecological assessment of OTC and Zn pollution. No earthworm mortality was observed, but neutral-red retention time and flow cytometry analyses showed severe damage to the integrity of the lysosomal membranes upon exposure to individual or combined pollutants. This damage might be a result of antibiotic-induced oxidative stress causing lipid peroxidation and reaction with cellular macromolecules, leading to DNA damage (Lin et al., 2012). On the other hand, heavy metals can induce the formation of active oxygen radicals, which would incite oxidative stress-related responses and cause lipid peroxidation, damaging cell membrane permeability (Wang et al., 2007). Lipid peroxidation can thus indirectly reflect the degree of intracellular damage (Wang et al., 2007). Baguer et al. (2000) tested the effects of OTC and tylosin on three species of soil fauna: Aporrectodea caliginosa (earthworms), Folsomia fimetaria (springtails), and Enchytraeus crypticus (enchytraeids). Neither of the substances had any biomarker-detectable effect at environmentally relevant concentrations. The lowest concentration observed to affect biomarkers was 3000 mg kg 1 and in many cases no effect was seen even at the highest experimental concentration of 5000 mg kg 1. Furthermore, Song et al. (2002) found that earthworm death did not occur until worms were exposed to concentrations of 1300 mg kg 1 Zn (LC50: 1500– 1900 mg kg 1). Compared with traditional monitoring of mortality, growth rate, and reproductive rate, lysosomal membrane stability and coelomocyte apoptosis are more sensitive, and are therefore appropriate biomarkers for identifying biological effects of lower-dose pollutants.
Table 2 Coelomocyte apoptosis rate of earthworms exposed to zinc (Zn) alone. Zn (mg kg
1
)
Q1 Q2 Q3 Q4
Coelomocyte apoptosis rate at 7 days (%)
Coelomocyte apoptosis rate at 14 days (%)
Coelomocyte apoptosis rate at 28 days (%)
CK
50
100
200
CK
50
100
200
CK
50
100
200
4.0 4.0 85 7.0
2.3 13 71 13
2.3 19 70 8.7
1.0 24 44 31
0.4 6.1 77 16
2.0 26 41 31
8.7 53 34 4.3
10 58 27 4.0
5.5 8.5 72 14
5.0 29 58 8.0
8.4 22 58 12
2.5 38 41 19
Q1: necrotic cells and impurities, Q2: late apoptotic cells, Q3: normal cells; Q4: early apoptotic cells.
Table 3 Coelomocyte apoptosis rate of earthworms exposed to oxytetracycline (OTC) alone (Gao et al., 2014). OTC (mg kg
Q1 Q2 Q3 Q4
1
)
Coelomocyte apoptosis rate at 7 days (%)
Coelomocyte apoptosis rate at 14 days (%)
Coelomocyte apoptosis rate at 28 days (%)
5
10
20
5
10
20
5
10
20
2.5 16 72 8.8
5.7 29 60 5.7
13 34 48 8.1
9.5 15 64 11
9.5 29 50 12
2.7 52 32 13
9.3 23 59 9.0
2.0 25 62 11
3.8 51 29 16
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M. Gao et al. / Chemosphere 139 (2015) 229–234 Table 4 Coelomocyte apoptosis rate of the earthworm exposed to combined pollution of oxytetracycline (OTC) and zinc (Zn). OTC + Zn (mg kg
Q1 Q2 Q3 Q4
1
)
Coelomocyte apoptosis rate at 7 days (%)
Coelomocyte apoptosis rate at 14 days (%)
Coelomocyte apoptosis rate at 28 days (%)
5 + 50
10 + 50
20 + 50
5 + 50
10 + 50
20 + 50
5 + 50
10 + 50
20 + 50
9.2 13 67 11
0.40 20 57 23
4.6 24 67 4.3
3.1 32 59 5.9
0.30 11 61 28
1.7 4.2 87 6.8
3.8 40 32 24
5.9 8.5 72 14
5.7 7.9 79 7.2
(a) Molecular structure
(b) Speciation diagram
Fig. 3. Molecular structure of OTC and speciation diagram of OTC in water (Han et al., 2009; Fig. 2).
Compared with Zn alone, NRRT inhibition and coelomocyte apoptosis rates under the combined pollutants were lower, indicating an antagonistic effect. Moreover, there was antagonism between OTC and Zn2+ with increasing exposure time. This indicates that OTC aggregation or complexation between OTC and Zn2+ might occur at a higher pH (8.0), leading to decreased toxicity. For example, Han et al. (2009) found that OTC sorption was promoted by an OTC–Zn–clay bridge between pH 5 and 9, and that the most significant effect occurred at pH 6.5. The sorption of Zn2+ on montmorillonite and kaolinite was decreased by OTC in the pH range 4–9. Either the cation competition of OTC at a lower pH or prohibition of sorption or precipitation resulting from complexation between OTC and Zn2+ at higher pH may explain this trend. Circular dichroism and UV–Vis spectrometry showed that complexation between OTC and a heavy metal could occur at the O11 or O12, O1 sites of OTC with increasing pH (Fig. 3). Tongaree et al. (1999) also observed OTC aggregation and metal–OTC complexation in solutions with Ca2+ and Mg2+ at pH 7.5. Additionally, zinc ions reduce the solubility of OTC because of zincate formation, which causes anionic OTC to precipitate. The zinc–OTC complex exhibited the highest crystallinity and lowest solubility at pH 8.0 (Tongaree et al., 1999). With increasing time, NRRT of the control decreased and early or late coelomocyte apoptosis increased. These observations could be attributed to the soil we used; although it was not contaminated with the target compounds, some other pollutants might have been present. The background values of Pb and Zn in soil were 8.2 and 8.3 mg kg 1, respectively. These concentrations might have been deleterious to lysosomal membranes and coelomocytes during incubation. These findings have clearly indicated that the neutral-red retention and coelomocyte apoptosis assays exhibit a sensitive, generalized response to pollutants, and, as such, are useful biomarkers for early warning of the individual and combined effects of contaminants in the soil.
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Moffett, B.F., Nicholson, F.A., Uwakwe, N.C., Chambers, B.J., Harris, J.A., Hill, T.C.J., 2003. Zinc contamination decreases the bacterial diversity of agricultural soil. FEMS Microbiol. Ecol. 43 (1), 13–19. Qu, M.M., Xu, Y., Chen, H., Li, Z., Sun, L., Xu, D., Kong, Z., Sugiura, N., 2005. Toxicological study of three veterinary drugs on Eisenia foetida. Chin. J. Appl. Ecol. 16 (6), 1108–1111. Rocco, A., Scott-Fordsmand, J.J., Maisto, G., Manzo, S., Salluzzo, A., Jensen, J., 2011. Suitability of lysosomal membrane stability in Eisenia fetida as biomarker of soil copper contamination. Ecotoxicol. Environ. 74, 984–988. Sforzini, S., Boeri, M., Dagnino, A., Oliveri, L., Bolognesi, C., Viarengo, A., 2012. Genotoxicity assessment in Eisenia andrei coelomocytes: a study of the induction of DNA damage and micronuclei in earthworms exposed to B[a]P and TCDD-spiked soils. Mutat. Res. 746 (1), 35–41. Song, Y.F., Xu, H.X., Ren, L.P., Song, C.X., Gong, P., 2003. Inhibition and eco-toxicity of heavy metals pollution on vegetable growth in soils. J. Agro-Environ. Sci. 22 (1), 13–15. Song, Y.F., Zhou, Q.X., Xu, H.X., Ren, L.P., Sun, T.H., Gong, P., 2002. Acute toxicological effects of heavy metal pollution in soils on earthworms. Chinese J. Appl. Ecol. 13 (2), 187–190. Svendsen, C., Meharg, A.A., Freestone, P., Weeks, J.M., 1996. Use of an earthworm lysosomal biomarker for the ecological assessment of pollution from an industrial plastics fire. Appl. Soil Ecol. 3, 99–107. Svendsen, C., Spurgeon, D., Hankard, P., Weeks, J., 2004. A review of lysosomal membrane stability measured by neutral red retention: is it a workable earthworm biomarker? Ecotox. Environ. Safe. 57 (1), 20–29. Thiele, S., 2005. Microbial inhibition by pharmaceutical antibiotics in different soils dose – effect relations determined with the iron (III) reduction test. Environ. Toxicol. Chem. 24 (4), 869–876. Tongaree, S., Flanagan, D.R., Poust, R.I., 1999. The interaction between oxytetracycline and divalent metal ions in aqueous and mixed solvent systems. Pharm. Dev. Technol. 4 (4), 581–591. Vanhala, P.T., Ahtiainen, J.H., 2006. Soil respiration, ATP content, and photobacterium toxicity test as indicators of metal pollution in soil. Environ. Toxicol. Water Qual. 9 (2), 115–121. Wang, L.P., Zhang, M.K., 2009. Effects of oxytetracycline pollution onsoil biological properties. J. Agro-Environ. Sci. 28 (7), 1434–1438. Wang, Q.L., Zhu, L., Huang, B.J., Li, Y., 2007. Toxicity effects of lead in polluted soil on earthworm coelomocyte lysosome. J. Agro-Environ. Sci. 26 (5), 1874–1878. Wu, S., Zhang, H., Zhao, S., Wang, J., Li, H., Chen, J., 2012. Biomarker responses of earthworms (Eisenia fetida) exposured to phenanthrene and pyrene both singly and combined in microcosms. Chemosphere 87 (4), 285–293. Yang, Z.X., Liu, S.Q., Zheng, D.W., Feng, S.D., 2006. Effects of cadium, zinc and lead on soil enzyme activities. J. Environ. Sci. 18 (6), 1135–1141. Yao, J.H., Niu, D.K., Li, Z.J., Liang, Y.C., Zhang, S.Q., 2010. Effects of antibiotics oxytetracycline on soil enzyme activities and microbial biomass in wheat rhizosphere. Sci. Agric. Sinica 43 (4), 721–728. Zhang, H.M., Zhang, M.K., Gu, G.P., 2008. Residues of tetracyclines in livestock and poultry manures and agricultural soils from north Zhejiang Province. J. Ecol. Rural Environ. 24 (3), 69–73. Zhang, S.Q., Zhang, F.D., Liu, X.M., Wang, Y.J., Zou, S.W., He, X.S., 2005. Determination and analysis on main harmful composition in excrement of scale livestock and poultry feedlots. Plant Nutr. Fert. Sci. 11 (6), 822–829.
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Biomarker analysis of combined oxytetracycline and zinc pollution in earthworms (Eisenia fetida) Minling Gao ⇑, Yun Qi, Wenhua Song, Qian Zhou Department of Environmental and Chemical Engineering, Tianjin Polytechnic University, No. 399 Binshui Western Road, Xiqing District, Tianjin 300387, China
h i g h l i g h t s exposure were proofed compared to Zn2+ alone. 2+ A mechanism involving complexation of Zn and OTC at alkaline pH is proposed. Lysosomal membrane stability and coelomocyte apoptosis are sensitive biomarkers. Antagonistic effects of OTC and Zn
a r t i c l e
2+
i n f o
Article history: Received 17 March 2015 Received in revised form 11 June 2015 Accepted 12 June 2015
Keywords: Coelomocyte apoptosis Earthworm Interactive effect Lysosomal membrane stability Antibiotic Heavy metal
a b s t r a c t To determine the interactive action of antibiotics and heavy metals, this study assessed pollutant-induced responses of cellular biomarkers in earthworms (Eisenia fetida) exposed to zinc (Zn2+) and oxytetracycline (OTC) in soil. Lysosomal membranes were damaged and coelomocyte apoptosis occurred with exposure to the individual and combined pollutants. Compared with Zn2+ alone, lysosomal membrane stability and coelomocyte apoptosis decreased in the Zn2+–OTC combined treatment, possibly as a result of complexation of Zn2+ and OTC at alkaline pH. Such complexation could reduce the toxicity of the pollutants. Lysosomal membrane stability and coelomocyte apoptosis are sensitive biomarkers and could be economical and rapid tools for the monitoring and assessment of a variety of pollutants. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The antibiotic oxytetracycline (OTC) is used extensively worldwide in veterinary drugs and feed additives, and is beginning to pollute soils. Most veterinary antibiotics cannot be completely absorbed by animals and are discharged through feces and urine as the parent compound or as metabolites (Halling-Søensen, 2001; Liao et al., 2001; Kong and Zhu, 2007). The impact of OTC on the environment is increasing with the development of intensive livestock and poultry breeding programs and the wide use of manure fertilizer. The average OTC content is as high as 9.1 mg kg 1 (1.1–135 mg kg 1) in pig manure and 6.0 mg kg 1 (2.9–23 mg kg 1) in chicken manure (Zhang et al., 2005). OTC concentrations averaged 5.2 mg kg 1 in surface soils (0–20 cm) from agricultural fields treated with animal manure in northern Zhejiang Province, China (Zhang et al., 2008). Hamscher et al. ⇑ Corresponding author. E-mail addresses: [email protected] (M. Gao), [email protected] (Y. Qi), [email protected] (W. Song), [email protected] (Q. Zhou). http://dx.doi.org/10.1016/j.chemosphere.2015.06.059 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
(2000) showed that contents of oxy- and chlortetracycline in surface soils (0–40 cm) were as high as 32 and 26 mg kg 1, respectively, after application of manure fertilizer. Tetracyclines, including OTC and other veterinary antibiotics, may lead to antibiotic resistance in microorganisms, which is potentially hazardous to non-target organisms in terrestrial environments. Various effects of OTC have been observed, including the inhibition of bacteria, actinomycetes, and total microorganism populations (Wang and Zhang, 2009); decreased urease, sucrose phosphatase, and hydrogen peroxidase activity (Yao et al., 2010); reduced root and shoot elongation in wheat and Chinese cabbage (Jin et al., 2009); and genotoxicity to earthworms (Dong et al., 2012). Zinc (Zn) is a trace element that is required for biological growth. The content of Zn in soil generally ranges from 10 to 300 mg kg 1 (Li et al., 2006), but Zn concentrations exceeding 13,500 mg kg 1 have been reported for polluted soil in China (Li et al., 2006). Some plants can absorb and accumulate Zn, but excess zinc uptake can inhibit plant growth and development (Song et al.,
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2003; Lin et al., 2007). Zinc can also affect soil respiration (Vanhala and Ahtiainen, 2006), enzyme activity (Yang et al., 2006; Lin et al., 2007), and bacterial diversity and population size (Moffett et al., 2003; Lu et al., 2010), and can have negative effects on soil and human health (Lock and Janssen, 2001; Jia et al., 2005). Antibiotics and heavy metals are often detected together in soils and organic fertilizers (Zhang et al., 2005). Combined antibiotic and heavy metal pollution has received increasing global attention. For example, metal- and antibiotic-resistant bacteria were found in Antarctic marine waters, and this was attributed to long-term exposure to low concentrations of these pollutants (De Souza et al., 2006). Ecotoxic effects of antibiotics in soil have been reported (Thiele, 2005; Bao, 2008; Hammesfahr et al., 2008; Liu et al., 2009). However, little information is available on the potential biochemical and genetic effects of veterinary antibiotics and heavy metals in soils, although a few studies have indicated that the combination of antibiotics and metals can be harmful to environmental bacteria and earthworms (Kong et al., 2006; Gao et al., 2013, 2014). More information on the effects of these pollutants is needed for improved soil risk assessment. Biomarkers in terrestrial invertebrates can be used to estimate the exposure to and effects of pollutants. Earthworms are sensitive indicators of soil quality and are widely used in terrestrial ecotoxicology (ISO, 1998; Lin et al., 2007). Many biomarkers have been utilized to profile the toxicological effects of pollutants, including lethality, LD50, reproductive rate, enzymatic activity, lysosomal membrane stability, cell apoptosis, and DNA damage (Sforzini et al., 2012; Wu et al., 2012). However, mortality, growth rate, and reproduction are commonly considered inappropriate for evaluating chemicals of lower toxicity and concentration. Qu et al. (2005) reported that OTC does not induce earthworm mortality at a concentration of OTC 500 mg/L. However, using the comet assay, these authors showed that OTC induced significant DNA damage in earthworm coelomocytes (P < 0.01). At the subcellular level, the stability of lysosomal membranes in coelomocytes can be used as a biomarker for the toxicological effects of contamination. One method that can be used to determine lysosomal membrane stability is the neutral-red retention assay. Neutral red retention time (NRRT) is a sensitive marker in earthworms that meets soil pollution risk assessment requirements (Svendsen et al., 2004; Jia et al., 2005). Flow cytometry accurately detects cell survival and apoptosis rates and reflects damage to cell membranes. This technique has been widely used to evaluate the toxicological effects of pollutants. Gao et al. (2014) reported a synergistic effect of 5 mg kg 1 OTC + 50 mg kg 1 lead (Pb) on earthworm lysosomes and they found antagonistic effects at higher concentrations (10–20 mg kg 1 OTC + 50 mg kg 1 Pb). In addition, coelomocyte apoptosis decreased significantly with combined OTC and Pb treatment compared with OTC alone, indicating an antagonistic reaction. In general, the modes of interactive effects between chemicals include antagonism, addition, and synergism. However, there is little information available on the interactive action of antibiotics and heavy metals. Knowledge of such interactions is important for assessing the toxicity, migration and transformation of pollutants, particularly compound pollution. Therefore, the objectives of this study were to (1) determine the interactive action of OTC and Zn2+ on earthworms, and (2) investigate the use of cellular biomarkers in earthworms (Eisenia fetida) exposed to zinc alone and in combination with OTC. We accordingly investigated lysosomal membrane stability and coelomocyte apoptosis for their potential use as sensitive, simple, and rapid biomarkers for soil monitoring and assessment.
2. Materials and methods 2.1. Soils and chemicals Surface soils (0–30 cm) were sampled from suburban farmland in Tianjin, China. The samples were air-dried, crushed to pass through a 10-mm-mesh screen, and stored in containers at 4 °C. The background contents of OTC and Zn were 0.0 and 8.3 mg kg 1, respectively. The soil had a medium loam texture with a density of 1.3 g cm 3; a pH of 8.2; an organic matter content of 1.6%; a water-holding capacity of 42%; a composition of 21% sand, 50% silt, and 29% clay; and a cation exchange capacity of 11 cmol kg 1. Oxytetracycline hydrochloride (95%–105% purity), EDTA, and dimethyl sulfoxide (DMSO, >99% purity) were obtained from Amresco (Ohio, USA). Guaiacol glyceryl ether (>98% purity) was purchased from Sigma (Beijing, China). Zinc nitrate (>99% purity) was obtained from Bodi Chemical Co. Ltd. (Tianjin, China). Lumbricus balanced salt solution (LBSS) consisted of 71.5 mM NaCl, 4.8 mM KCl, 3.8 mM CaCl2, 1.1 mM MgSO47H2O, 0.4 mM KH2PO4, 0.3 mM Na2HPO47H2O, and 4.2 mM NaHCO3. 2.2. Earthworms Earthworms (E. fetida) were purchased from Tianjin Chenggong Earthworm Farm Company, China. The worms were preincubated in clean soil for 1 week under controlled conditions (22 ± 2 °C, 12 h light:12 h dark, 75% humidity) before the experiment. Adult earthworms with a developed clitellum were collected for testing; the individual wet weight of adults ranged from 300 to 500 mg. 2.3. Experimental design Zinc nitrate at a concentration of 0, 50, 100, or 200 mg kg 1 was added to beakers containing 500 g soil. Oxytetracycline hydrochloride at a concentration of 5, 10, or 20 mg kg 1 was added to beakers containing 500 g soil and 50 mg kg 1 Zn. These dosages were chosen based on reported concentrations of OTC and Zn in soil. Moisture content was adjusted to 60% of the water-holding capacity. All tests were performed in triplicate. The soil was allowed to stabilize for 24 h prior to earthworm introduction. Ten worms were placed in each beaker and the beakers were enclosed with plastic film perforated with small ventilation holes. The test was conducted under a photoperiod of 12 h light:12 h dark at 20 ± 2 °C for 28 days. Humidity was maintained at 75% ± 5% by an artificial climate chamber. Worms were fed rewetted oven-dried cow manure (0.5 g per worm) every 7 days thereafter over the 28-day period. In order to maintain the original moisture percentage, distilled water was added gravimetrically to the replicates every week. Two earthworms were collected from each beaker without replacement at 7, 14, and 28 days. Although there was no earthworm death during the experiment, there was a significant difference in body weight loss among the different groups of earthworms. 2.4. Harvesting of coelomocytes Earthworms were allowed to empty their guts for 18 h in Petri dishes on filter paper moistened with distilled water. Each worm was rinsed with distilled water and saline (6.5 mg mL 1 NaCl, 4 °C) before being placed into a 1.5-mL Eppendorf tube containing 1 mL cold extrusion medium for 2 min. The extrusion solution contained 5% ethanol, 95% saline, 2.5 mg mL 1 EDTA, and 10 mg mL 1 guaiacol glyceryl ether and was adjusted to pH 7.3 with 1 M NaOH. Earthworms were removed from the tubes and the cell suspension
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was set aside for 10 min. Part of the cell-containing supernatant (0.5 mL) was transferred to another tube, and combined with LBSS. After centrifugation (100g and 4 °C for 3 min), the precipitate containing cells was collected and suspended in distilled water, adjusted to pH 7.3 with NaOH (Goven et al., 1993). Recovered cells were centrifuged at 100g and 4 °C for 3 min and then re-suspended in LBSS. Cell viability (trypan blue exclusion method) was assessed immediately after extrusion and was approximately 90% in all cases.
2.5. Neutral-red retention time Lysosomal membrane stability was assessed according to Svendsen et al. (1996). Briefly, 20-lL coelomic fluid samples were placed on a microscope slide and the cells were allowed to adhere to the glass surface for 30 s. The slides were then stained with 20 lL neutral red working solution (80 mg mL 1), which contained 2.5 mL LBSS and 10 lL neutral red stock solution. The neutral red stock solution was freshly prepared by dissolving 20 mg neutral red powder in 1 mL DMSO. The working solution was renewed every hour to avoid crystallization. Cover slides were placed and the slides were observed every 2 min under a microscope at 400; the slides were maintained in the artificial climate chamber between observations. Observations were stopped when the ratio of cells with fully stained cytosol was greater than 50% of the total number of cells counted. This time is referred to as the neutral red retention time.
7d 14d 28d
NRRT/min
50 40 30
** **
20
**
10
** ** **
** ** **
0 ck
50
100
Coelomocyte samples were washed twice with cold phosphate-buffered saline and then were suspended repeatedly with 200 lL 1 binding buffer. Subsequently, 100-lL aliquots of cell suspension were transferred to 1.5-mL Eppendorf tubes containing 10 lL Annexin V-PE. After mixing by inversion, the tubes were placed in an ice bath for 20–30 min in the dark. Subsequently, 380 lL 1 binding buffer and 10 lL 7-AAD dye were added to the cell suspensions and mixed gently. Finally, the cell samples were analyzed using a FACS Calibur flow cytometer (BD Biosciences, USA). 2.7. Statistical analysis To evaluate the effect of chemical exposure on earthworms, the NRRT inhibition rate was calculated as follows: NRRT inhibition rate = (NRRTcontrol group NRRTexposure group)/NRRTcontrol group. All data are presented as means ± standard deviation (SD). Significant differences between treatments were analyzed by one-way analysis of variance (ANOVA) using SPSS version 16.0 (SPSS, Inc., Chicago, IL). 3. Results 3.1. Combined effects of OTC and Zn on lysosomal membrane stability Compared to the control, the NRRT of coelomocyte lysosomal membranes decreased significantly (P < 0.01) after 7 days of exposure to Zn, and this affect increased with increasing Zn concentration (P < 0.05) (Fig. 1). Zinc exposure had a similar effect on lysosomal membranes after 14 and 28 days, and the effect was concentration dependent and highly significant (P < 0.05) at 200 mg kg 1. The NRRT inhibition rate at days 14 and 28 was 72% ± 5.6% and 80% ± 3.1%, respectively (Table 1). In contrast, NRRT decreased with increasing incubation time. The lowest inhibition rates occurred on day 14 and were 52% ± 9.4% (Zn = 50 mg kg 1), 68% ± 4.0% (Zn = 100 mg kg 1), and 72% ± 5.2% (Zn = 200 mg kg 1) (Table 1). Membrane stability decreased significantly in worms exposed to the combination of OTC and Zn (P < 0.05). Compared to Zn alone, exposure to combined OTC and Zn enhanced the NRRT inhibition rate with increasing incubation time (P < 0.05) but generally decreased the NRRT inhibition rate as OTC concentration increased (Fig. 2). The NRRT inhibition rate increased by 19%, 43%, and 78% at the dose of 5 mg kg 1 OTC + 50 mg kg 1 Zn, 10 mg kg 1 OTC + 50 mg kg 1 Zn and 20 mg kg 1 OTC + 50 mg kg 1 Zn at day 7 compared to the inhibition rate by Zn alone. Moreover, the
70 60
2.6. Flow cytometry
200
Zn (mg/kg) Fig. 1. Neutral red retention time of the earthworm exposed to single Zn.
Table 1 Combined effect of zinc (Zn) and oxytetracycline (OTC) on neutral red retention time (NRRT) inhibition rate. Time (days) 7
14
28
OTC concentration (mg kg 1)
NRRT inhibition rate (%)
Zn (mg kg
CK 5 10 20
58 ± 5.0 35 ± 4.1 54 ± 1.4 84 ± 1.4
CK 5 10 20 CK 5 10 20
NRRT inhibition rate (%)
OTC + Zn concentration (mg kg 1)
NRRT inhibition rate (%)
Combined effect
50 100 200
65 ± 6.2 69 ± 1.5 78 ± 3.1
5 + 50 10 + 50 20 + 50
81 ± 7.9 77 ± 11 71 ± 3.4
Antagonism Antagonism Antagonism
36 ± 4.5 53 ± 4.6 60 ± 5.5 75 ± 5.5
50 100 200
52 ± 9.4 68 ± 4.0 72 ± 5.6
5 + 50 10 + 50 20 + 50
80 ± 4.3 68 ± 11 43 ± 8.6
Antagonism Antagonism Antagonism
19 ± 4.0 42 ± 9.6 48 ± 5.8 67 ± 4.4
50 100 200
64 ± 9.1 72 ± 5.2 80 ± 3.1
5 + 50 10 + 50 20 + 50
72 ± 14 77 ± 14 61 ± 3.1
Antagonism Antagonism Antagonism
1
)
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4. Discussion
70
7d 14d 28d
60
NRRT/min
50 40 30
** 20
** 10
** ** ** * *
** **
**
0 ck
5+50
10+50
20+50
OTC+Zn (mg/kg) Fig. 2. Neutral red retention time of the earthworm exposed to combined pollution of OTC and Zn.
NRRT inhibition was strongest after 14 days of exposure to OTC + Zn; it was increased by 25%, 43%, and 83% at the dose of 5 mg kg 1 OTC + 50 mg kg 1 Zn, 10 mg kg 1 OTC + 50 mg kg 1 Zn and 20 mg kg 1 OTC + 50 mg kg 1 Zn compared to that under the corresponding Zn-only treatments (Table 1). 3.2. Combined effects of OTC and Zn on coelomocyte apoptosis Coelomocyte apoptosis in earthworms increased significantly in the Zn-only treatments (P < 0.05) (Table 2). Early and late apoptosis increased with increasing Zn concentration after 7 days of exposure and reached maximums of 31% and 24%, respectively, at 200 mg kg 1 Zn (Table 2). Late apoptosis generally increased (P < 0.01) with increasing Zn concentration after 14 days of exposure and reached 58% at 200 mg kg 1 Zn. After 28 days of exposure, early and late apoptosis rates were 19% and 38%, respectively (Table 2). Compared to Zn alone, coelomocyte apoptosis was significantly higher in earthworms exposed to OTC + Zn (P < 0.05) and the effects were dependent on OTC concentration (Table 4). However, coelomocyte apoptosis rates under the combined pollutants were lower than the sums of Zn-only and OTC-only (Table 3).
Although earthworm biomarker responses to heavy metals (Bundy et al., 2007; Annamaria Rocco et al., 2011; Calisi et al., 2013) and organic chemicals (Calisi et al., 2011) have previously been profiled, few studies have examined the effects of organic pollutants and heavy metals in combination. Earthworms provide an early warning of adverse environmental conditions (Calisi et al., 2013). The effects of pollutants at the cellular or subcellular level should occur before, or at lower concentrations than, effects at the population, community, or ecosystem scale (Svendsen et al., 1996). Thus, cellular dynamics are increasingly examined when evaluating the effects of xenobiotic pollutants on soil organisms. In the present study, lysosomal membrane stability and coelomocyte apoptosis were examined as cell biomarkers for the ecological assessment of OTC and Zn pollution. No earthworm mortality was observed, but neutral-red retention time and flow cytometry analyses showed severe damage to the integrity of the lysosomal membranes upon exposure to individual or combined pollutants. This damage might be a result of antibiotic-induced oxidative stress causing lipid peroxidation and reaction with cellular macromolecules, leading to DNA damage (Lin et al., 2012). On the other hand, heavy metals can induce the formation of active oxygen radicals, which would incite oxidative stress-related responses and cause lipid peroxidation, damaging cell membrane permeability (Wang et al., 2007). Lipid peroxidation can thus indirectly reflect the degree of intracellular damage (Wang et al., 2007). Baguer et al. (2000) tested the effects of OTC and tylosin on three species of soil fauna: Aporrectodea caliginosa (earthworms), Folsomia fimetaria (springtails), and Enchytraeus crypticus (enchytraeids). Neither of the substances had any biomarker-detectable effect at environmentally relevant concentrations. The lowest concentration observed to affect biomarkers was 3000 mg kg 1 and in many cases no effect was seen even at the highest experimental concentration of 5000 mg kg 1. Furthermore, Song et al. (2002) found that earthworm death did not occur until worms were exposed to concentrations of 1300 mg kg 1 Zn (LC50: 1500– 1900 mg kg 1). Compared with traditional monitoring of mortality, growth rate, and reproductive rate, lysosomal membrane stability and coelomocyte apoptosis are more sensitive, and are therefore appropriate biomarkers for identifying biological effects of lower-dose pollutants.
Table 2 Coelomocyte apoptosis rate of earthworms exposed to zinc (Zn) alone. Zn (mg kg
1
)
Q1 Q2 Q3 Q4
Coelomocyte apoptosis rate at 7 days (%)
Coelomocyte apoptosis rate at 14 days (%)
Coelomocyte apoptosis rate at 28 days (%)
CK
50
100
200
CK
50
100
200
CK
50
100
200
4.0 4.0 85 7.0
2.3 13 71 13
2.3 19 70 8.7
1.0 24 44 31
0.4 6.1 77 16
2.0 26 41 31
8.7 53 34 4.3
10 58 27 4.0
5.5 8.5 72 14
5.0 29 58 8.0
8.4 22 58 12
2.5 38 41 19
Q1: necrotic cells and impurities, Q2: late apoptotic cells, Q3: normal cells; Q4: early apoptotic cells.
Table 3 Coelomocyte apoptosis rate of earthworms exposed to oxytetracycline (OTC) alone (Gao et al., 2014). OTC (mg kg
Q1 Q2 Q3 Q4
1
)
Coelomocyte apoptosis rate at 7 days (%)
Coelomocyte apoptosis rate at 14 days (%)
Coelomocyte apoptosis rate at 28 days (%)
5
10
20
5
10
20
5
10
20
2.5 16 72 8.8
5.7 29 60 5.7
13 34 48 8.1
9.5 15 64 11
9.5 29 50 12
2.7 52 32 13
9.3 23 59 9.0
2.0 25 62 11
3.8 51 29 16
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M. Gao et al. / Chemosphere 139 (2015) 229–234 Table 4 Coelomocyte apoptosis rate of the earthworm exposed to combined pollution of oxytetracycline (OTC) and zinc (Zn). OTC + Zn (mg kg
Q1 Q2 Q3 Q4
1
)
Coelomocyte apoptosis rate at 7 days (%)
Coelomocyte apoptosis rate at 14 days (%)
Coelomocyte apoptosis rate at 28 days (%)
5 + 50
10 + 50
20 + 50
5 + 50
10 + 50
20 + 50
5 + 50
10 + 50
20 + 50
9.2 13 67 11
0.40 20 57 23
4.6 24 67 4.3
3.1 32 59 5.9
0.30 11 61 28
1.7 4.2 87 6.8
3.8 40 32 24
5.9 8.5 72 14
5.7 7.9 79 7.2
(a) Molecular structure
(b) Speciation diagram
Fig. 3. Molecular structure of OTC and speciation diagram of OTC in water (Han et al., 2009; Fig. 2).
Compared with Zn alone, NRRT inhibition and coelomocyte apoptosis rates under the combined pollutants were lower, indicating an antagonistic effect. Moreover, there was antagonism between OTC and Zn2+ with increasing exposure time. This indicates that OTC aggregation or complexation between OTC and Zn2+ might occur at a higher pH (8.0), leading to decreased toxicity. For example, Han et al. (2009) found that OTC sorption was promoted by an OTC–Zn–clay bridge between pH 5 and 9, and that the most significant effect occurred at pH 6.5. The sorption of Zn2+ on montmorillonite and kaolinite was decreased by OTC in the pH range 4–9. Either the cation competition of OTC at a lower pH or prohibition of sorption or precipitation resulting from complexation between OTC and Zn2+ at higher pH may explain this trend. Circular dichroism and UV–Vis spectrometry showed that complexation between OTC and a heavy metal could occur at the O11 or O12, O1 sites of OTC with increasing pH (Fig. 3). Tongaree et al. (1999) also observed OTC aggregation and metal–OTC complexation in solutions with Ca2+ and Mg2+ at pH 7.5. Additionally, zinc ions reduce the solubility of OTC because of zincate formation, which causes anionic OTC to precipitate. The zinc–OTC complex exhibited the highest crystallinity and lowest solubility at pH 8.0 (Tongaree et al., 1999). With increasing time, NRRT of the control decreased and early or late coelomocyte apoptosis increased. These observations could be attributed to the soil we used; although it was not contaminated with the target compounds, some other pollutants might have been present. The background values of Pb and Zn in soil were 8.2 and 8.3 mg kg 1, respectively. These concentrations might have been deleterious to lysosomal membranes and coelomocytes during incubation. These findings have clearly indicated that the neutral-red retention and coelomocyte apoptosis assays exhibit a sensitive, generalized response to pollutants, and, as such, are useful biomarkers for early warning of the individual and combined effects of contaminants in the soil.
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