Investigating the effects of vermicomposting process using Eisenia Fetida earthworms on the reduction of parasites population

Abstract

Purpose

The existence of parasite agents in natural organic fertilizers can lead to health problems and infection transmission. The aim of the present study was to survey the parasites’ population reduction during the vermicomposting of the mixtures of municipal sewage sludge (SS) and cow dung (CD) using E. fetida earthworms.

Methods

The vermicomposting process was performed by using earthworms of E. fetida species. The composting process was conducted in 27 pilots for 3 months. The identification and counting of the parasites’ population were carried out by Mac Master Slide, according to Bailenger method.

Results

The results indicated that the type and number of parasite elements decreased with increasing vermicomposting time. The cumulative removal percent of parasites for sewage sludge (96.10%) was more than the SS + CD (93.65%) and CD (92.93%) treatments. The results showed that after 90 days of vermicomposting, the highest cumulative reduction in the number of parasites was obtained for the treatments with 40 earthworms (98.48%), while the corresponding value for the treatments without earthworms was (88.66%). The statistical analysis indicated that in terms of the parasite’s population, a significant difference was observed for the three kinds of compostable materials (P < 0.05), however, this difference was not significant for the three levels of the earthworms (P > 0.05).

Conclusion

The results showed that the mixture of sewage sludge and cow dung in combination with sawdust could lead to greater and faster reduction in the parasite’s population. In addition, the bio-transformed product can be used as useful manure.

Introduction

Biosolids such as cow dung, agricultural residues, household wastes, and sewage sludge are good sources of organic substances and plant nutrients [1]. Nowadays, the use of these biosolids, as manure in agriculture, is a common practice in some parts of the world, specifically in rural areas of developing countries [2–4]. Also, the use of the other organic wastes such as municipal and domestic sewage sludge as fertilizer is increasing dramatically due to limiting traditional sources of organic matter such as animal manure [5, 6]. Due to health and environmental problems caused by the excessive use of chemical fertilizers, the application of organic fertilizers and compost has been recommended instead of them seriously [7]. One of the most important and determinant factors in the quality of compost and sewage sludge is the type and the amount of pathogenic microbes and parasites [8]. Many factors including the state of health of the population, the presence of hospitals, tanneries, meat-processing factories and abattoirs in the same area can affect the type of pathogens, which are most commonly found in sewage sludge. Pathogenic microorganisms such as bacteria, viruses, protozoa and eggs of parasitic agents, such as Oocyte, D. dendriticum eggs, E. histolytica cyst, Ascaris lumbricoides eggs, etc. can be found in sewage sludge [9]. Several studies have reported that the vermicomposting process under specific and controlled processing conditions can reduce the population of parasites to a safe level [10]. Generally, those parasites that their eggs or larvae are discharged into the environment with excrements are considered for investigation. The United States Environmental Protection Agency (U.S. EPA) has reported that the pathogens in domestic wastewater are primarily associated with insoluble solids. In wastewater treatment plants, primary wastewater treatment processes concentrate these solids into sewage sludge via the practice of settling and adsorption [11]. As a result, the untreated or raw primary sewage sludges can have higher quantities of pathogens than the incoming raw wastewater. Most of wastewater treatment processes cannot completely remove and destroy microorganisms available in the incoming raw wastewater and the generated sewage sludge. Therefore, pathogenic microorganisms, especially the parasites eggs, can enter into the environment through the effluent or the resulting biological sewage sludge [11]. It has been reported that more than 40 million people in the world are infected with parasitic infections and more than 10% of the world’s population are exposed to the risk of infection resulting from parasitic diseases [12]. Thus, improper disposal or management of wastewaters, sludges, and cattle manures are among the most important resources that spread infective agents, such as parasites, into the environment. Subsequently, these organic materials can cause serious environmental pollutions due to their putrescible characteristics [13–15]. Furthermore, the production of industrial and domestic sludges, as by-products of wastewater treatment plants, has been increasing drastically. It can be majorly due to growing urbanization, the demand for better water quality, and the imposition of stricter environmental laws [16]. Nowadays, the treatment and disposal of the generated sludges are considered as one of the most critical environmental issues of wastewater treatment plants both in developed and developing countries; so that about half, even up to 60%, of the total wastewater treatment costs is associated with these processes [16–18]. Since sewage sludge is categorized as hazardous waste materials, therefore, selecting or identifying a simple and appropriate method or technology, with low capital, operational, and maintenance costs, to reduce and stabilize these kinds of biosolids is very important and is a high priority [2, 17]. Among various physical, chemical, and biological technologies, which have been developed to manage biodegradable wastes such as sewage sludge and organic solid wastes, composting or a combination of composting-vermicomposting is considered as a suitable, promising, and eco-friendly method for the management of biodegradable wastes [17, 19, 20]. Vermicomposting is an advantageous organic waste conversion technology, in which the joint action of earthworms and microorganisms decomposes and stabilizes biodegradable raw materials [17, 21]. Although, microbes are responsible for biochemical degradation of organic matter during vermicomposting, earthworms are the most important drivers of the process, because they amend the substrate and alter the microbiological activity [17, 22]. Earthworms also have the ability to bioaccumulate heavy metals, Polycyclic Aromatic Hydrocarbons (PAHs), and toxins [23–25]. Additionally, the end product of the vermicomposting process has high contents of nitrogen, phosphorous, and humic acids as well as low human pathogens. These characteristics made it suitable to be used as a soil amendment or conditioner. It also improves the physical, chemical, and microbial properties of the soil, and stimulates the growth of plants [16, 17]. In addition, selling of the earthworms can lead to revenue generation, indicating that vermicomposting can be more economical than the conventional composting process [21]. It has been reported in the literature that some earthworm species (based on their ecological niche) have the ability to consume a wide range of organic wastes such as sewage sludge, cattle dung, crop residues, and industrial refuse. Among the various groups of earthworms that have been evaluated in composting technology, the epigamic category of earthworms, including E. fetida species, are the best candidate for vermicomposting [21, 22]. These species are widely used throughout the world for the management of organic wastes, due to their voracious feeding habit, wider acceptability of organic waste materials, and high fecundity [21, 22]. By considering the above facts, firstly, the present study was carried out to investigate the potential of E. fetida in the vermicomposting of different mixtures of the municipal sewage sludge and cow dung to produce a suitable fertilizer. Secondly, the reduction in the numbers or population of the pathogenic parasites was also investigated.

Material and methods

Substrates and earthworms

This experimental research was performed in a pilot scale. Sewage sludge (SS) and cow dung (CD) were used as raw materials, and sawdust was used as a bulking agent for vermicomposting. Sewage sludge was collected randomly after biological treatment from a municipal wastewater treatment plant in Yathrib district of Ghaemshahr City, Mazandaran province, Iran. The sludge was dewatered in gunny bags and was air-dried before use. Fresh cow dungs were collected from a cattle farm in a village around of Neka City, Mazandaran province, Iran, and were kept at 4 °C for further use. The earthworms of E. fetida (a number of 1200 worms) were manually obtained from the soil and cow manure in the north of Iran. All the earthworms were placed in a mixture of cow dung and sewage sludge to feed, adopt, grow, and increase their population.

Experimental design

The sample size was estimated by a full factorial method using the Design Expert 7 software. Vermicomposting was carried out in 27 plastic pots with 2 kg capacity. Initial mixtures were prepared with dewatered sewage sludge (SS) and cow dung (CD) (weight ratio of 50:50). The sawdust (as the bulking agent) was added, with the ratio in the range of 7.5–15%, to the dewatered sewage sludge and cow dung. To adjust the C/N ratio at the optimum range, the mixtures containing 100% sewage sludge and the mixtures containing 50% sewage sludge and 50% cow dung were respectively mixed with 15% and 7.5% (weight ratio) sawdust.

At first, 4 cm of fine sand and then 4 cm of loam soil were added to the plastic pot to prepare suitable bedding for the earthworms’ activities. The compost production process was performed on 1.2 kg of the mixtures of sewage sludge and cow dung (at three levels 0, 50, and 100 wt%) and E. fetida (at three numbers of 0, 20, and 40) in 27 pilots for 3 months. Optimum conditions for moisture (60–80%) and temperature (10–30 °C) were maintained and controlled during the process.

Parasitological analyses

The amount of 4 g homogenized and free of earthworms samples were picked up from each plastic container on the 0st, 30th, 60th, and 90th days of vermicomposting. Then, these samples were transferred to the parasitology laboratory of the school of medicine, Mazandaran University of Medical Sciences, for identification of parasites, according to the standard methods [26]. Totally, 108 samples were gathered from all the pilots for determination of parasites. In briefly, all samples were solved in 10 cc sterile distilled water and kept for 2 h at the room temperature. Large particles were deposited. The supernatant was discarded and the pellet was centrifuged twice at 1000 rpm for 15 min.

After settling, the supernatant was extracted and acetoacetic buffer with the formula CH₃COCH₂COOH (pH = 4.5) and acetylcholine (ether) with equal volume were added to each centrifuge tube. The tubes were shaken several times and the samples were completely mixed by stirring and, consequently, were centrifuged for third times at 1000 rpm for 15 min. The samples were analyzed using the Bailenger method by Mc Master counting slide (with a hole size 0.3 ml) according to the U.S. EPA method 1600 [27].The deposited material was suspended in zinc sulfate 33% (with 5 volumes and density of 1.18) and completely mixed by stirring. Finally, 0.3 ml of this solution was taken and moved to the McMaster slide with 100 and 40 magnifications. The quantity of the cysts and parasite eggs was recognized and counted by microscope (Olympus Microscope CX21, China). The number of helminth eggs or cysts in each sample was calculated through the following equation:

$$N=\frac{\mathit{AX}}{\mathit{PV}}$$ 1

where N is the number of helminth eggs or cysts per 4 g of the sample, A is the average number of parasite eggs or cysts, which were counted on the three slides under microscopic observation, X is the final volume of the product (mL), P is the volume of pot on the McMaster slide (3 mL), and V represents the wet weight of the original sample (4 g).

Statistical analyses

Statistical analyses were performed using the SPSS software (version 22.0; SPSS Inc., Chicago, IL). The association between the number and type of the helminth eggs or cysts for the three treatments (SS + CD, SS, and CD) was measured by ANOVA using the generalized linear model (GLM) and non-parametric method of Kruskal–Wallis tests. In addition, the number of worms was estimated using non-parametric method of Kruskal–Wallis test. The mean data of these variables in the end product of compost were compared to the different compost standards such as Iranian national (class 1and 2), WHO and U.S. EPA (class A and B) standards.

Results

Effects of the kind of compostable material on the parasite population

The changes in the average number and the type of parasites identified (per 4 g wet wt of the sample) for different feeding substrates are shown in Table 1 and Fig. 1. It is obvious from Fig. 1(a, b, and c) that the fresh substrates or feeding materials contained considerable parasites loads. As presented in the table, the average number of all parasites decreased with increasing the vermicomposting time for all the three treatments (SS + CD, SS, and CD). For instance, the population of all identified parasites on 0 day of vermicomposting of the mixture of sewage sludge and cow dung (SS + CD treatment) was 439, while the corresponding value on 90th day of the process was 27.8. A same trend was also found for the other treatments (SS and CD). The results of the ANOVA test revealed that there was statistically significant difference among feeding substrates or treatments with respect to the average number of parasites (P < 0.001). The substrates also showed significant differences for parasites population at different vermicomposting times (T₀, T₃₀, T₆₀ and T₉₀) (P < 0.001). On the initial day (T₀), there were 8, 8, and 11 types of parasites in SS + CD, CD, and SS, while on 90th day (T₉₀) 6, 5, and 6 kinds of parasites were detected and identified at the same conditions, respectively (Table 1). On the initial day (T₀), in CD treatment, the highest and the lowest number of parasites was related to E. coli cyst and Tenia echinococcus egg, respectively. Ascaris lumbricoides egg (in SS + CD and CD) and E. coli cyst (in SS) had the highest number. Rabed tiofaed larvae without bulb (in SS + CD and CD), Rabed tiofaed larvae with bulb (in SS + CD, SS and CD), Oocyte, D. dendriticum egg, F. hepatica egg, Trichostrongylus egg (in SS), and Tenia echinococcus egg (in CD) had the lowest average numbers of parasites on 90th day (T₉₀). It can be seen from the table that the average number of all parasites decreased as the vermicomposting time increased. It is worth noting that in some treatments some parasites such as D. dendriticum egg, Blastocystis hominis, F. hepatica egg, G. lamblia cysts, Trichostrongylus egg, and Tenia echinococcus egg were not detected during vermicomposting process. More details about the average number, the type of the identified parasites, and the reduction rate during the vermicomposting are presented in Table 1. The mean cumulative reduction in the average number of parasite for Ascaris lumbricoides egg was 82.7%, and the corresponding value for Rabed tiofaed larvae without bulb, and Rabed tiofaed larvae with bulb was 100% in SS + CD treatment. In SS treatment, the corresponding values for Ascaris lumbricoides egg was 78.81%, and for Oocyte, D. dendriticum egg, Hookworms egg, F. hepatica egg, Rabed tiofaed larvae with bulb, and Trichostrongylus egg, it was 100%. Furthermore, in CD treatment, the reduction rate was 81.75% for E. coli cyst and 100% for Rabed tiofaed larvae without bulb, Rabed tiofaed larvae with bulb, and Tenia echinococcus egg. After 90 days of the process (T₉₀), 96.10% and 92.93% of all the parasites population was reduced in SS and CD treatments, respectively.

Table 1.

The number and type of parasites during various vermicomposting times for different raw compostable materials (treatments)

Parasite name	Time (days)	SS + CD Treatment	SS Treatment	CD Treatment	Cumulative reduction (%)			Significance (P value)
		Mean ± SD	Mean ± SD	Mean ± SD	SS + CD	SS	CD	Material	Time
Oocyte	0 (beginning)	55.82 ± 17.04	80.67 ± 31.97	NS	–	–	NS	< 0.001	<0.001
	30	37.20 ± 12.75	50.38 ± 28.28	NS	33.36	37.55	NS
	60	19.08 ± 12.51	15.98 ± 16.09	NS	65.82	80.19	NS
	90	5.76 ± 7.63	00.00 ± 00.00	NS	89.68	100	NS
D. dendriticum egg	0 (beginning)	NS	67.32 ± 12.74	NS	NS	–	NS	<0.001	<0.001
	30	NS	46.10 ± 12.75	NS	NS	31.53	NS
	60	NS	8.98 ± 9.90	NS	NS	86.66	NS
	90	NS	00.00 ± 00.00	NS	NS	100	NS
E. histolytica cyst	0 (beginning)	46.18 ± 13.68	NS	75.54 ± 16	–	NS	–	<0.001	<0.001
	30	26.68 ± 9.43	NS	56.64 ± 16.8	42.23	NS	25.02
	60	11.64 ± 8.26	NS	20.96 ± 15	74.79	NS	72.25
	90	2.62 ± 4.35	NS	5.20 ± 9.33	94.33	NS	93.12
E. coli cyst	0 (beginning)	74.06 ± 18.27	72.08 ± 17.42	86.12 ± 10	–	–	–	=0.405	<0.001
	30	51.23 ± 13.10	53.66 ± 15.24	51.02 ± 11.6	30.83	25.56	40.76
	60	27.35 ± 14.44	33.54 ± 12.56	35.48 ± 12.8	63.07	53.47	58.80
	90	8.98 ± 10.28	9.24 ± 10.37	15.72 ± 15.7	87.87	87.18	81.75
Ascaris lumbricoides egg	0 (beginning)	53.40 ± 8.62	62.09 ± 11.51	42.14 ± 7.26	–	–	–	<0.001	<0.001
	30	38.73 ± 6.78	46.12 ± 8.96	27.12 ± 5.50	27.47	25.73	35.64
	60	23.62 ± 5.57	28.40 ± 8.04	13.42 ± 5.18	55.77	54.26	68.15
	90	9.24 ± 6.13	13.16 ± 7.67	3.66 ± 3.93	82.70	78.81	91.31
Hookworms egg	0 (beginning)	51.78 ± 16.62	77.76 ± 17.89	39.64 ± 7.29	–	–	–	=0.001	<0.001
	30	27.65 ± 11.00	35.90 ± 10.13	22.50 ± 6.34	46.60	53.83	43.24
	60	6.13 ± 5.72	8.26 ± 10.34	10.06 ± 7.94	88.16	89.38	74.62
	90	0.62 ± 1.86	00.00 ± 00.00	3.66 ± 4.66	98.80	100	90.77
Blastocystis hominis	0 (beginning)	NS	NS	44.20 ± 10.7	NS	NS	–	<0.001	<0.001
	30	NS	NS	25.66 ± 9.18	NS	NS	41.95
	60	NS	NS	5.38 ± 5.41	NS	NS	87.83
	90	NS	NS	0.62 ± 1.86	NS	NS	98.60
Free –living fagellate	0 (beginning)	54.82 ± 13.12	70.77 ± 18.63	36.68 ± 6.91	–	–	–	=0.019	<0.001
	30	27.36 ± 9.06	26.72 ± 13.89	24.44 ± 8.62	50.09	62.25	33.37
	60	4.96 ± 6.35	7.18 ± 10.30	9.38 ± 8.18	90.95	89.86	74.43
	90	0.62 ± 1.86	1.84 ± 3.99	1.88 ± 2.82	98.87	97.40	94.87
F. hepatica egg	0 (beginning)	NS	52.89 ± 12.83	NS	NS	–	NS	<0.001	<0.001
	30	NS	12.86 ± 8.90	NS	NS	75.69	NS
	60	NS	00.00 ± 00.00	NS	NS	100	NS
	90	NS	00.00 ± 00.00	NS	NS	100	NS
Rabed tiofaed larvae without bulb	0 (beginning)	58.30 ± 17.31	85.78 ± 19.15	56.90 ± 14.2	–	–	–	=0.002	<0.001
	30	21.06 ± 10.13	30.66 ± 24.69	13.64 ± 10.7	63.88	64.26	76.03
	60	2.43 ± 3.81	7.96 ± 11.73	3.46 ± 7.64	95.83	90.72	100
	90	00.00 ± 00.00	0.66 ± 1.98	00.00 ± 00.0	100	99.23	100
Rabed tiofaed larvae with bulb	0 (beginning)	44.34 ± 10.31	66.40 ± 16.00	29.86 ± 7.59	–	–	–	<0.001	<0.001
	30	17.71 ± 7.08	26.18 ± 13.18	7.62 ± 7.05	60.06	60.58	74.48
	60	1.40 ± 4.20	2.60 ± 5.19	0.66 ± 1.98	96.84	96.08	97.79
	90	00.00 ± 00.00	00.00 ± 00.00	00.00 ± 00.0	100	100	100
G. lamblia cysts	0 (beginning)	NS	48.14 ± 11.41	NS	NS	–	NS	<0.001	<0.001
	30	NS	25.14 ± 14.09	NS	NS	47.78	NS
	60	NS	9.50 ± 12.99	NS	NS	80.27	NS
	90	NS	2.64 ± 5.95	NS	NS	92.44	NS
Trichostrongylus egg	0 (beginning)	NS	48.03 ± 13.07	NS	NS	–	NS	<0.001	<0.001
	30	NS	15.08 ± 8.97	NS	NS	68.60	NS
	60	NS	00.00 ± 00.00	NS	NS	100	NS
	90	NS	00.00 ± 00.00	NS	NS	100	NS
Tenia echinococcus egg	0 (beginning)	NS	NS	23.98 ± 4.35	NS	NS	–	<0.001	<0.001
	30	NS	NS	6.78 ± 4.69	NS	NS	71.73
	60	NS	NS	1.26 ± 2.50	NS	NS	94.75
	90	NS	NS	00.00 ± 00.0	NS	NS	100
Total number of parasite	0 (beginning)	438.70 ± 99.3	731.96 ± 79.8	435 ± 24.7	–	–	–	<0.001	<0.001
	30	274.62 ± 56.9	368.80 ± 93.8	235.4 ± 49.8	35.56	49.62	45.89
	60	96.61 ± 49.14	122.40 ± 77.7	100 ± 42.5	77.98	83.28	77.80
	90	27.84 ± 27.76	28.54 ± 23.74	30.74 ± 25.3	93.65	96.10	92.93

Values indicate mean ± standard deviation are based on nine samples (n = 9). SS = sewage sludge, CD = cow dung and SS + CD = the mixture of sewage sludge and cow dung

All values are given per 4 g wet weight

NS  Not Seen

Fig. 1.

The changes in the average number and the type of parasites in different treatments at various vermicomposting times (a:SS + CD, b: SS, c: CD)

Effects of the number of earthworms on parasite population

Our results indicated that the cumulative reduction ratio for each identified parasite enhanced with increasing the number of earthworms (Table 2). The mean total number of parasites reduced over time and with an increase in the number of earthworms. The results of the between subjects 2-way ANOVA and Kruskal–Wallis tests revealed that no significant difference in the mean number of parasites was observed with an increase in the number of the earthworms at four different times (T₀, T₃₀, T₆₀ and T₉₀) (P = 0.097). On the initial day (T₀), 14 types of parasites such as Oocyte, D. dendriticum egg, E. histolytica cyst, E. coli cyst, A. lumbricoides egg, hookworms egg, B. hominis, free–living flagellate, F. hepatica egg, R. tiofaed larvae without bulb, R. tiofaed larvae with bulb, G. lamblia cysts, Trichostrongylus egg, and T. echinococcus egg were detected and identified for each of the three levels of worms (W₀, W₂₀, and W₄₀). On 90th day (T₉₀), there were 9, 6 and 3 types of parasites in W₀, W₂₀, and W_40, respectively (Table 2)_.

Table 2.

The number and types of parasites during vermicomposting for various levels of earthworms

Parasite name	Time (days)	Without worm (W0)	20 worms (W20)	40 worms (W40)	Cumulative reduction (%)			Significance (P value)
		Mean ± SD	Mean ± SD	Mean ± SD	W0	W20	W40	Worms	Time
Oocyte	0 (beginning)	44.72 ± 36.23	47.68 ± 43.26	44.09 ± 44.27	–	–	–	=0.663	<0.001
	30	34.68 ± 28.87	31.62 ± 31.89	21.28 ± 23.2	22.45	33.68	51.74
	60	20.07 ± 18.08	10.37 ± 11.38	4.62 ± 7.14	55.12	78.25	89.52
	90	5.18 ± 8.87	0.58 ± 1.74	00.00 ± 00.0	88.42	98.78	100
D. dendriticum egg	0 (beginning)	23.72 ± 36.47	23.20 ± 35.49	20.40 ± 3	–	–	–	=0.898	<0.001
	30	18.82 ± 29.12	15.52 ± 23.53	11.76 ± 18	20.66	33.10	42.37
	60	7.10 ± 10.96	1.26 ± 2.50	0.62 ± 1.86	70.07	94.57	96.96
	90	00.00 ± 00.00	00.00 ± 00.00	00.00 ± 00	100	100	100
E. histolytica cyst	0 (beginning)	43.06 ± 39.67	41.56 ± 35.11	37.10 ± 29.6	–	–	–	=0.553	<0.001
	30	32.44 ± 32.46	29.26 ± 26.67	21.62 ± 18.5	24.66	29.60	41.73
	60	18.56 ± 17.32	10.54 ± 9.66	3.50 ± 4.56	56.90	74.64	90.57
	90	7.24 ± 9.09	0.58 ± 1.74	00.00 ± 00	83.19	98.60	100
E. coli cyst	0 (beginning)	73.18 ± 11.97	75.48 ± 21.68	83.60 ± 13.4	–	–	–	=0.110	<0.001
	30	57.67 ± 7.30	46.40 ± 13.45	51.84 ± 15.38	21.19	38.53	37.99
	60	44.44 ± 9.01	28.67 ± 9.68	23.26 ± 11.1	39.27	62.02	72.18
	90	21.90 ± 12.40	9.40 ± 10.26	2.64 ± 4.44	70.07	87.55	96.84
Ascaris lumbricoides egg	0 (beginning)	51.24 ± 9.36	51.70 ± 11.67	54.69 ± 15.9	–	–	–	= 0.793	<0.001
	30	38.25 ± 9.00	35.76 ± 10.19	37.96 ± 13.2	25.35	30.83	30.60
	60	24.42 ± 8.29	20.46 ± 8.10	20.56 ± 10.40	52.34	60.43	62.41
	90	13.26 ± 8.26	7.68 ± 4.87	5.12 ± 5.66	74.12	85.15	90.64
Hookworms egg	0 (beginning)	50.14 ± 11.18	56.96 ± 25.51	62.08 ± 25.4	–	–	–	=0.663	<0.001
	30	32.63 ± 6.09	29.40 ± 13.33	24.02 ± 10.5	34.92	48.38	61.31
	60	16.53 ± 6.29	5.32 ± 5.50	2.60 ± 4.35	67.03	90.66	95.81
	90	2.98 ± 4.70	0.68 ± 2.04	0.62 ± 1.86	94.06	98.81	99.00
Blastocystis hominis	0 (beginning)	14.72 ± 23.07	13.22 ± 20.78	16.26 ± 24.7	–	–	–	= 0.960	<0.001
	30	9.80 ± 15.64	7.52 ± 12.35	8.34 ± 13.33	82.34	87.75	92.87
	60	2.60 ± 5.16	1.62 ± 3.32	1.16 ± 3.48	95.79	100	100
	90	0.62 ± 1.86	00.00 ± 00.00	00.00 ± 00.0	0.00	0.00	0.00
Free –living fagellate	0 (beginning)	55.96 ± 18.38	55.82 ± 22.04	50.49 ± 19.4	–	–	–	=0.018	<0.001
	30	34.70 ± 7.78	26.26 ± 8.69	17.56 ± 7.30	37.99	52.96	65.22
	60	16.63 ± 5.86	3.53 ± 4.48	1.36 ± 4.08	70.28	93.68	97.31
	90	3.66 ± 3.98	0.68 ± 2.04	00.00 ± 00.0	93.46	98.78	100
F. hepatica egg	0 (beginning)	16.64 ± 25.09	15.16 ± 24.11	21.09 ± 32.1	–	–	–	=0.936	<0.001
	30	6.76 ± 10.49	3.36 ± 6.75	2.74 ± 6.09	59.38	77.84	87.01
	60	00.00 ± 00.00	00.00 ± 00.00	00.00 ± 00.0	100	100	100
	90	00.00 ± 00.00	00.00 ± 00.00	00.00 ± 00.0	100	100	100
Rabed tiofaed larvae without bulb	0 (beginning)	61.42 ± 24.89	67.36 ± 19.28	72.20 ± 20.2	–	–	–	=0.304	<0.001
	30	36.90 ± 19.29	19.08 ± 11.80	9.38 ± 6.44	39.92	71.67	87.01
	60	9.20 ± 11.30	1.19 ± 2.36	00.00 ± 00.0	85.02	98.23	100
	90	0.66 ± 1.98	00.00 ± 00.00	00.00 ± 00.0	98.93	100	100
Rabed tiofaed larvae with bulb	0 (beginning)	45.44 ± 18.55	47.40 ± 18.33	47.76 ± 22.3	–	–	–	=0.542	<0.001
	30	24.81 ± 14.54	15.84 ± 9.48	10.86 ± 7.35	45.40	66.58	77.27
	60	4.66 ± 5.86	00.00 ± 00.00	00.00 ± 00.0	89.74	100	100
	90	00.00 ± 00.00	00.00 ± 00.00	00.00 ± 00.0	100	100	100
G. lamblia cysts	0 (beginning)	16.52 ± 25.07	13.30 ± 21.24	18.32 ± 27.8	–	–	–	=0.548	<0.001
	30	13.64 ± 20.64	6.00 ± 9.76	5.50 ± 10.21	17.43	54.89	69.99
	60	8.88 ± 13.33	00.00 ± 00.00	0.62 ± 1.86	46.25	100	96.62
	90	3.64 ± 5.95	00.00 ± 00.00	00.00 ± 00.0	77.97	100	100
Trichostrongylus egg	0 (beginning)	16.90 ± 25.90	14.96 ± 22.75	16.17 ± 26.6	–	–	–	=0.921	<0.001
	30	7.68 ± 11.83	5.20 ± 8.45	2.20 ± 4.75	54.56	65.24	86.39
	60	00.00 ± 00.00	00.00 ± 00.00	00.00 ± 00.0	100	100	100
	90	00.00 ± 00.00	00.00 ± 00.00	00.00 ± 00.0	100	100	100
Tenia echinococcus egg	0 (beginning)	7.94 ± 11.99	8.04 ± 12.40	8.00 ± 12.35	–	–	–	=0.912	<0.001
	30	3.44 ± 5.42	1.88 ± 4.03	1.46 ± 2.90	56.68	76.62	81.75
	60	1.26 ± 2.50	00.00 ± 00.00	00.00 ± 00.0	84.13	100	100
	90	00.00 ± 00.00	00.00 ± 00.00	00.00 ± 00.0	100	100	100
Total number of parasite	0 (beginning)	521.60 ± 146	531.84 ± 164	552.29 ± 183	–	–	–	=0.097	<0.001
	30	352.22 ± 94.9	273.10 ± 75.8	226.52 ± 54	32.47	48.65	58.99
	60	174.35 ± 39.6	82.96 ± 21.38	58.30 ± 18.1	66.57	84.40	89.44
	90	59.14 ± 13.02	19.69 ± 12.57	8.38 ± 7.03	88.66	96.31	98.48

On 90th day of vermicomposting process (T₉₀)_, E. histolytica cyst (in W₀), E. coli cyst (in W₂₀), A. lumbricoides egg (in W₄₀) had the highest number of parasite. However, D. dendriticum egg, F. hepatica egg, R. tiofaed larvae with bulb (in W₀, W₂₀ and W₄₀), B. hominis, R. tiofaed larvae without bulb, G. lamblia cysts, Trichostrongylus egg, T. echinococcus egg (in W₂₀ and W₄₀), and Oocyte, E. histolytica cyst and free-living flagellate (in W₄₀) contained the lowest numbers of parasites.

The lowest and highest cumulative reduction of parasites during the process in the treatment with no earthworms (W₀) were as follows: E. coli cyst (70.07%), and D. dendriticum egg, F. hepatica egg, R. tiofaed larvae with bulb, Trichostrongylus egg, and T. echinococcus egg (100%). In treatment W₂₀, the corresponding cumulative reduction for each parasite was as follows: A. lumbricoides egg (85.15%) and 100% for D. dendriticum egg, F. hepatica egg, R. tiofaed larvae without bulb, R. tiofaed larvae with bulb, G. lamblia cysts, Trichostrongylus egg, and T. echinococcus egg. In treatment W₄₀, 100% reduction was observed for Oocyte, D. dendriticum egg, E. histolytica cyst, free –living Flagellate, F. hepatica egg, R. tiofaed larvae without bulb, R. tiofaed larvae with bulb, G. lamblia cysts, Trichostrongylus egg, and T. echinococcus egg, and the reduction value for A. lumbricoides egg was 90.64%. It is apparent from the table that the highest and lowest cumulative reduction in the number of parasites was obtained for W₄₀ (98.48%) and W₀ (88.66%), respectively, after 90 days of vermicomposting (end day or T₉₀). The total number of parasites in the final product of the vermicomposting process was compared to the upper limit values of guidelines recommended by the national organization (standards class 1 and 2), WHO, and U.S. EPA (class A and B) for the application of compost in agriculture (Table 3).

Table 3.

Comparison of total number of parasites at the end product of the vermicomposting process with compost standards

Characteristics					Vermicomposting produced from different kinds of compostable materials			Vermicomposting produced from different numbers of worms
					Mixture of sewage sludge + cow dung	Sewage sludge	Cow dung	0 (without) Worm	20 Worm	40 Worms
Total number of parasite elements (No 4 g−1 wet wt)	Result of this study				27.84	28.54	30.74	59.14	19.69	8.38
	Compost standards	Iran	Class 1	< 1	*	*	*	*	*	*
			Class 2	–	–	–	–	–	–	–
		U.S. EPA	Class A	< 1	*	*	*	*	*	*
			Class B	–	–	–	–	–	–	–
		WHO		< 1	*	*	*	*	*	*

*: Asymmetrical

Discussion

In line with other studies, in the present research, the mean number of parasites was decreased with an increase in the vermicompost processing time for all pilot tests. In the study conducted by Karimi et al. [10] on the changes in microbial pathogen dynamics during vermicomposting, the same results were also obtained. In contrast with our results, Singh and Suthar [28] reported that the fungi population increased during vermicomposting. This difference may be related to differences in the methodology, raw materials, weight of the samples, test conditions, duration of study, the species of earthworms, and type and amount of compostable materials [28]. On the other hand, the reduction of parasites during composting and vermicomposting process can be due to the influence of temperature and the enzymes secreted by the earthworms [28]. Generally, the parasite reduction in the SS treatment was greater, compared to CD and SS + CD treatments. The number and type of parasites were decreased not only by the activity of the earthworms and microorganisms during vermicomposting process, but also the addition of sawdust as the bulking agent to the compostable materials can play an effective role in the reduction of the parasite elements [29]. Hashemimajd and Jamaati-e-Somarin [30] indicated that the mixing of a suitable proportion of the bulking material and the sewage sludge with the ratio of 30:30 (V/V) improved the stability of the sewage sludge produced in the sewage treatment plants. Parvaresh et al. [31] indicated that the number of total microorganisms decreased to A class standards of U.S.EPA at the end of the process. Our results showed that this composting method is reliable and simple to operate, with high flexibility and low odor production. The amount of organic compounds and the number of pathogenic microorganisms were reduced during this study and the final product had an acceptable quality. Amouei et al. [19] described that the quality of the compost produced from the mixed materials was in line with the U.S. EPA class A standards. Dominguez et al. [32] and Panikkar et al. [33] demonstrated that the number of microorganisms reduced during the vermicomposting process carried out by different species of earthworms. According to the present study, the number and type of parasites reduced for the three levels of worms (W₀, W_20, and W₄₀). This reduction was more significant in W₄₀ in comparison with the other two levels. The worms number is an essential factor for vermistabilization/vermicomposting of all solid wastes [34].

The reduction in A. lumbricoides egg was less than the other type of parasites; indicating that it was the most resistant parasite egg during the vermicomposting process. Our results indicated that some of soil parasites such as R. tiofaed larvae (soil nematode) and other soil protozoa were seen during all stages, and their removal percentage was changed slowly over time. The parasitic quality of the compost produced by using the earthworms was better than the compost produced without the use of the earthworms. The use of E. Fetida species in the compostable materials produced compost with better quality in terms of the number of parasites and heavy metals [29]. Our results indicated that the reduction in the number of parasites was high in the SS + CD, and especially, in the SS treatments. This reduction represented the role of E. Fetida species and the application of the bulk materials such as sawdust. The results presented in Table 3 indicated that the total number of parasites at the end product of vermicomposting process is not close to the U.S. EPA standards. In T₃₀, the reduction percent of parasites in CD was greater than the mixture of SS + CD treatment, but at the other times (T₆₀ and T₉₀) the amounts of parasites decreased more strongly in the mixture of SS + CD than CD.

In addition to the three factors such as time, type of compostable material, and the addition of sawdust as bulking agent, which had a significant role in the reduction of the amount of parasites, the use of a higher number of the earthworms also had a positive effect on the reduction of the number of parasites during the vermicomposting process. This process moved forward faster, with the high ratio of decreasing in the number of parasites.

Generally, at T₃₀, T₆₀, and T₉₀ of vermicomposting process, the reduction of different types of parasites in the SS + CD treatment was slower than the other compostable materials (SS and CD) (P > 0.05). In the present study, the parasites such as beef Ascaris eggs, eggs of F. hepatica, larvae paramsy, soil nematode, and many protozoa were lost during the vermicompost process. In the studies conducted by Yousefi et al. (2004), Omrani et al. (1973), and Graff et al. (1953), the similar results were also obtained [35–37].

Conclusions

Vermicomposting is a simple technology requiring low capital, operational, and maintenance costs, which can be used for bioconversion of organic matter. Earthworms have the ability to both increase the rate of aerobic degradation and composting of organic matter present in the sewage sludge and also to stabilize organic residues by removing harmful agents such as parasite pathogens. Our results indicate that the use of vermicomposting process can be used as an alternative technology for the reduction of parasitic pathogens. The addition of accessible and low-cost bulking agent, such as sawdust, alongside with the application of the earthworms during the vermicomposting process of sewage sludge mixed with cow dung can lead to a superior and faster reduction in the parasites population.

Declarations

Conflict of interests

The authors declare that they have no conflict of interest.