1. Introduction
Due to limited fossil energy resources, renewable bio-energy technologies are regarded as alternative of fossil fuels in the future [
1,
2]. In recent years, biogas based on methane fermentation is becoming an attractive energy resource in many nations worldwide [
3]. In China, there are more than 1.5 × 10
4 biogas projects based on livestock and poultry breeding [
4], which was expected to alleviate the energy shortage and reduce the emission of the waste.
However, it still poses environmental risk in biogas project even slurry was successfully anaerobically digested. Zhong
et al. found there were high concentrations of heavy metals, such as Cd Cu, Ni, and Zn in the digested slurry [
5]. In addition, some toxic organic pollutants also influence the effective utilization of biogas slurry, such as polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated biphenyls (PCB), polyaromatic hydrocarbons (PAH), perfluorinated alkyl compounds (PFCs), linear alkylbenzene sulfonates (LASs), nonylphenols and nonylphenol ethoxylates (NP/NPEOs) and polybrominated diphenyl ethers (PBDEs) [
6]. Recent research has focused on treatment methods for slurry, which could be potential requirement of the risk evaluation of slurry, especially toxicity.
On the other hand, some useful toxicity evaluation methods have been applied to the industrial effluents. Recent study finds the treated olive mill wastewater still had a certain degree of toxicity to
Vibrio fischeri [
7]. Similarly, it was possible that post-treated slurry would be still not suitable to release to river although it had been treated and reach discharge standard. Thus, biological toxicity tests
in vivo and
in vitro need to be applied to slurry management.
Bulich, A.A.
et al. firstly reported the possibility that
Phototobacterium phosphoreum could be applied to the toxicity evaluation of wastewater in the 1800s [
8], more and more model organisms were developed. Up to now, more than eight methods have been developed and applied to different industrial effluents. Main bio-indicators could be luminescent bacteria [
9], algae [
10],
Daphnia magna [
11,
12] and fish.
In these methods, the luminescent bacterium test, or Microtox test is one of the assays that frequently used for the acute toxicity assessment of environmental samples such as water and sediments [
13], as well as pure compounds [
14], for its relatively inexpensive, well-reproducible results, and fast testing procedure. The luminescence of bacteria depends on the existence of Adenosine triphosphate (ATP), fluorescein (FMN) and luciferase, so any factors that interfere or damage the respiration of the bacteria or physiological process can reduce the activity of luminescent bacteria. There are many different kinds of luminescent bacteria, such as
Vibrio,
Photobacterium,
Shewanella,
Xetorhabdus, and the most of
Vibrio,
Photobacterium,
Shewanella were marine bacteria. So far,
Photobacterium phosphoreum,
Vibrio fischeri (ISO 11348-3) [
15], and
Vibrio qinghaiensis were widely used into total toxicity evaluation of different industrial effluent, such as textile finishing industry [
16], tannery wastewaters [
17] and so on.
For further evaluation of health risk, toxicity tests with early development stage of aquatic organisms have been introduced as a faster and more cost-effective way. Moreover, study showed that the early developmental stages of fish are often the most sensitive to toxic effects [
18]. For relatively large, robust embryos and rapid embryonic development, zebrafish could be an ideal vertebrate model organism; moreover, transparent body is easily observed when zebrafish is developing outside their mother fish [
19,
20,
21]. It was reported that some indexes, such as embryos production, atretic oocytes and altered ovarian histology and embryos mortality, could be useful to evaluate pharmaceutical mixture and municipal wastewater [
22].
In this study, biological toxicity testing method was introduced to pig slurry, where related report on toxicity of slurry is still lack of study now. Here, V. fischeri, newly hatching larvae of zebrafish was used to evaluate acute toxicity of pig slurry, and embryos (1 hpf, 1 h post-fertilization) of zebrafish was for development toxicity. Using these methods, both digested and post-treated slurry were investigated to provide useful information about health risk of slurry and ability of the treatment process to reduce the toxicity of pig slurry, combined with physicochemical indexes (chemical oxygen demand (COD), ammonia nitrogen (NH3-N), total phosphorus (TP), etc.).
In this study, biological toxicity testing method was introduced to pig slurry, where related report on toxicity of slurry is still lack of study now. Here, V. fischeri, newly hatching larvae of zebrafish was used to evaluate acute toxicity of pig slurry, and embryos (1 hpf, 1 h post-fertilization) of zebrafish was for development toxicity. Using these methods, both digested and post-treated slurry were investigated to provide useful information about health risk of slurry and ability of the treatment process to reduce the toxicity of pig slurry, combined with physicochemical indexes (chemical oxygen demand (COD), ammonia nitrogen (NH3-N), total phosphorus (TP), etc.).
2. Experimental Section
2.1. Collection and Determination of Physicochemical Index of Samples
All samples of pig slurry were collected in Xinxin Forage Corporation in Jiaxing City of China. And before, pig slurry was pretreated by solid-liquid separation. Anaerobically digested and post-treated slurry (a 43 L membrane bio-reactor) were collected respectively. Digested slurry was collected in the outlet of anaerobic digester. Treated slurry was collected in the outlet of membrane bioreactor. Each index was determined three times respectively. After collection, physic-chemical variables were determined. Conductivity, pH, NH
3-N, TP and COD were determined by conductivity meter (Monitoring and analysis method of water and waste water, in Chinese), glass electrode method (GB/T6920-1986, in Chinese), nessler’s reagent colorimetric method (GB/T7479-1987, in Chinese), ammonium molybdate spectrophotometric method (GBT11893-1989, in Chinese) and potassium dichromate method (GB/T11914-1989, in Chinese) respectively. And then the samples were stored at 4 °C until used. Before the toxicity experiments, all samples were diluted, where de-ion water was used in luminescence experiment, and standard dilution water [
23] was used in zebrafish experiment.
2.2. Luminescent Bacteria
Freeze-dried marine luminescent bacteria (V. fischeri NRRL B-11177) were made in ampoule using freeze-drier (FD-5/8., Beijing Boyikang Test Co., Beijing, China). After the recovery of the freeze-dried powder, the initial luminous intensity needed to be between 2 × 106 and 5 × 106, which was detected by GloMax-Multi Detection System (Promega Co., Wisconsin, WI, USA) with a 96-well microplate (Corning/Costar Co., New York, NY, USA).
2.3. Zebrafish
Adult zebrafish (AB strain) were maintained in a recirculating aquaculture system (Aquaneering Co., San Diego, CA, USA). In incubation process, the 12 h light period was followed 12 h dark period per day. In the light period, the fish were fed with freshly hatched shrimp eggs and flake fish food (Tetra, Melle, Germany), twice and once respectively. The incubation temperature was controlled at 28 ± 0.5 °C.
2.4. Fertilized of Zebrafish Embryos
To hatch zebrafish embryos, one adult female fish and one adult male fish were placed in the same box. After the formation of zygote, embryos were washed 2–3 times by standard dilution water, for removing residues. Finally, normal developed fertilized eggs which were observed by the TS100-F microscope (Nikon, Tokyo, Japan) were collected for subsequent experiments.
2.5. Toxicity Tests
2.5.1. Luminescent Bacteria Toxicity Test
Luminescent bacteria test was performed using 96-well microplate on the GloMax-Multi Detection System. Due to high toxicity of slurry and in order to eliminate the effect of the color and density on results, filtered samples were diluted to avoid complete inhibition. In this paper, volume percentage of sample in de-ion water was adopted to represent dilution degree, where raw sample is 100%
v/v and de-ion water which was used as control sample was 0%
v/v. The procedure in detail which was referring to research of Froehner
et al. [
24] was as follows: 100 μL de-ion water as blank controls was added to the first row of microplate, 100 μL sample with various dilution(respectively 100%, 50%, 25%, 12.5%, 6.25%,
v/v) were added to the second, third, fourth, fifth and sixth row of microplate respectively. And then 100 μL bacteria suspension were added to each test well. After 15 min exposure, the luminescence intensity was measured. All of tests were repeated three times, while average luminescence intensity was adopted to dose-effect plot. Finally, the toxicity of slurry was characterized by relative luminous intensity and the concentration for 50% of maximal effect (EC
50).
2.5.2. Larvae of D. Rerio Acute Toxicity Test
The
D.
rerio 96 h acute toxicity test was carried out according to the procedure described in ISO7346-1 [
25]. To detect toxicity using zebrafish larvae, digested and post-treated slurry were diluted to a series of exposure solutions to avoid complete inhibition due to high toxicity of slurry. Here volume percentage of sample in standard dilution water was adopted to represent dilution degree, where raw sample of digested and post-treated slurry is 100%
v/v and standard dilution water which was used as control sample was 0%
v/v. In exposure experiment, ten normally developed larvae were transferred to each culture dish (100 mm) containing 15 mL sample with different dilution degree. The number of dead larvae was counted at 24 h, 48 h, 72 h and 96 h after exposure to slurry. In this experiment, five different dilution degrees were introduced to evaluate the toxicity of slurry.
2.5.3. Toxicity Test of Zebrafish Embryos
To detect toxicity using zebrafish embryos, diluted samples were needed to be prepared to avoid complete inhibition due to high toxicity of slurry. Normal embryos (at approximately 1 hpf) were kept in 24 well cell culture plates, with one embryo per well. Each well contained 1ml control or exposure wastewater. Two replicates for the controls and exposure groups were used. For each control and exposure group, the early embryonic development was observed by the TS100-F microscope and mortality was recorded at an interval of 24 h. After 72 h exposure experiments, mortality, hatching rate and malformation rate of embryos in each group were recorded. Similarly, five different dilution degrees were adopted [
26].
2.6. Methods of Toxicity Evaluation
The toxicity of pig slurry on zebrafish larvae was evaluated using 96 h lethal concentration 50 (LC
50), for
V.
fischeri and zebrafish embryos, EC
50 and ELC
50, HEC
50 and MEC
50 were used respectively. In this paper, ELC
50, HEC
50 and MEC
50 were used to represent the concentration of wastewater for 50% of embryonic mortality, hatching and malformation respectively while exposed to the pig slurry. Finally, toxicity unit (TU) was used to represent the toxicity directly. TU was calculated according to the formula as follows [
27]:
If the inhibition of luminescence intensity for
V.
fischeri, hatching rate for zebrafish embryos, mortality for zebrafish larvae were lower than 50% and malformation rate for zebrafish embryos was lower than 50% exposed to pig slurry, it showed LC
50 (EC
50) couldn’t be calculated, TU was calculated according to the formula as in Equation (2) [
27]:
where RE was the relative inhibition rate of
V.
fischeri luminosity and death rate of larvae and embryos of zebrafish (%)
And the toxicity remove rate was calculated according to the formula as follows:
2.7. Statistical Analysis
Using origin 8.0 (Origin Lab, Northampton, MA, USA), the median lethal concentration (LC50) and the median effect concentration (EC50) on D. rerio and luminescent bacteria of pig slurry were calculated, followed with one-way ANOVA in SPSS 16.0. Here statistical significance difference of exposure group to the control group was set to p < 0.01.
4. Discussion
The application of biogas engineering has received considerable attention in recent years, while there are many controversies about the advantages and disadvantages of biogas slurry. In some studies, slurry could be used to give rise of high yields to crops.J. Abubaker
et al. [
29] conducted a study on the fertilizing performance of pig slurry and mineral fertilizer in terms of spring wheat yield, in conclusion, pig slurry gave the overall highest yields to wheat. Gobernaa
et al. [
30] compared biogas digestates with fresh manure to the inhibition of pathogenic bacteria in soils, and found anaerobic digestion significantly sanitized the manure by completely eliminating cultivable
E.
coli and
Salmonella. However, toxic pollutants in the biogas slurry could be dangerous to environment. Many different pollutants had been found in slurry. For evaluation of availability of slurry, some researches were conducted. Using
Daphnia magna, A.I. De la Torre
et al. [
31] stated that toxicities of pig slurry were higher than urban effluents and lower than industrial effluents.
For evaluation of acute and development toxicity of slurry,
V.
fischeri, zebrafish larvae and embryos were exposed to digested and post-treated slurry respectively. In this study, different trophic level organisms showed different sensitivity to digested and post-treated slurry. The sensitivity order of the three test organisms was larva > embryo >
V.
fischeri. The difference of larva and embryo of zebrafish could result from membrane out of the embryo, which selectively restrict big molecule compounds to enter into embryo, which could be regarded as protective effect on the embryo [
32], as had been reported by Wiegand C
et al. [
33,
34]. There is little literature on the conductivity requirements for zebrafish larva and embryo, but adult zebrafish are tolerant to conductivity ranging from 400 μS∙cm
−1 to more than 1000 μS∙cm
−1 [
35]. In this study, conductivity of digested and post-treated slurry was both far higher than 1000 μS∙cm
−1. In addition, there was a correlation between conductivity and salinity generally. And
V.
fischeri was regarded as marine bacteria, so probably the higher sensitivity of fish versue
V.
fischeri could be mainly due to the different salinity tolerance. It showed the use of the marine bacteria tests is very important because it can avoid effect of salinity on test organism, this conclusion has also been confirmed by Pardo
et al. [
36].
Figure 4.
Variance analysis for relative luminosity of V. fischeri exposed to digested and post-treated slurry. Data are mean standard deviation; n = 3, per group. ** p < 0.01, others were no significant differences.
Figure 4.
Variance analysis for relative luminosity of V. fischeri exposed to digested and post-treated slurry. Data are mean standard deviation; n = 3, per group. ** p < 0.01, others were no significant differences.
Result also demonstrated that pig slurry had a high toxicity to both
V.
fischeri and zebrafish. The toxicity of digested slurry to
V.
fischeri was 14.68 TUs, which was higher than the TU of phenol (12.5 TUs) and lower than that of dimethyldiuron (16.23 TUs). Meanwhile, the toxicity of post-treated slurry to
V.
fischeri was 0.80 TU, which was between the TU of polyethylene glycol (0.78 TU) and cypermethrin (0.91 TU) [
37]. In addition, relative luminosity of
V.
fischeri exposed to post-treated slurry, was very significantly inhibited comparing with the de-ion water. On the other hand, relative luminosity of
V.
fischeri was higher than 100% (ANOVA,
p < 0.01;
Figure 4), when effluent was diluted to a certain degree of volume percentage. Some nutrients existed in pig slurry could enhance the cell activity of luminescent bacteria. Similar results could be observed in pure compounds experiments. For example, relative luminosity of
V.
fischeri was higher than 100% when the concentration of phenol was lower than 0.005 mg/L [
38]. Probably because that the inhibition on biological effect was lower, and bacterial recovery effect is stronger.
In addition, zebrafish larvae were all dead when exposed to digested and post-treated slurry, the same effect as the zebrafish embryos exposed to digested slurry, however the mortality of embryos exposed to post-treated slurry was lower than 50%. Meanwhile, effects of pig slurry on the early developing zebrafish embryos were observed. The experiment of embryos exposed to post-treated slurry also showed that the hatching rate was lower than 30% and malformation rate was more than 40%. According to reported development toxicity test of urban sewages using zebrafish embryos, hatching rate of embryos was more than 80% and malformation rate of embryos was lower than 20% respectively [
39]. It showed the toxicity of pig slurry was higher than urban sewages.
Moreover, 96 h LC
50 and toxicity of post-treated slurry to the zebrafish larvae were higher than 70% and lower than 2 TUs respectively. According to acute toxicity test of zebrafish larvae exposed to fat-plant effluent, showed that the value of LC
50 of larvae exposed to effluent was lower than 70%, reported by Şişman T.
et al. [
40]. In addition, adult zebrafish were used to evaluate the toxicity of industry effluent, such as electronic and electroplate effluent, the result showed the TU of effluent was 2.54 [
41]. Then considering that zebrafish larva is more sensitive than adult zebrafish. Thus, toxicity of post-treated slurry was lower than industrial effluents. And it showed that it was necessary to apply treatment to slurry, owing to high environmental risk.
Apparently, we could find the toxicity of digested slurry was reduced mostly after the treatment of MBR according to the luminescent bacteria toxicity test and zebrafish larvae acute toxicity test, the removal rate was 94.6% and 97.5% respectively, meanwhile according to the results of physicochemical index determination, post-treated slurry could be discharged legally. However, mortality of larvae still reached 100% when exposed to post-treated slurry. Besides other toxics, such as ammonia could be responsible for the toxicity of pig slurry. Recent study shows that ammonia can be physiologically harmful to
Hypophthalmichthys molitrix larvae, through increasing the concentration of reactive oxygen species and oxidative damage products such as lipid peroxides [
42]. Among the physiochemical characteristics of the slurry (NH
3-N, TP and COD), there was a high correlation between the toxicity removal rate and reduction rate of NH
3-N. It meant the decrease of concentration of NH
3-N, toxicity of pig slurry has also been reduced. So result that NH
3-N made much greater contribution on slurry toxicity could be obtained and further treatment should be used in pig slurry disposal or reused of final effluent.
In this paper, risk information was acquired while evaluation method was applied to pig slurry. However, more organisms for quantitative assessment are necessary. So a battery of bioassays based on organisms belong to different trophic levels, are strongly suggested, which could get a risk score by the application of a synthetic index for toxicity [
43], combining algae,
Daphnia magna and so on.