Nativa, Sinop, v. 11, n. 2, p. 241-250, 2023.

Pesquisas Agrárias e Ambientais

DOI: https://doi.org/10.31413/nativa.v11i2.15905

ISSN: 2318-7670

Effect of the balanced and nano-composite NPK fertilizer on the vitality of

the earthworm

Octolasion cyanieum

and its effect on the histological

composition of the middle of the body

Dhafar Hassan MOHAMMED1* , Adnan Mosa MOHAMMED1,

1 Department of Biology, College of Education for Pure Sciences, University of Mosul, Mosul, Iraq.

*E-mail: dhafar.21esp57@student.uomosul.edu.iq; dr.adnanmosa@uomosul.edu.iq

Submitted on 07/11/2023; Accepted on 08/04/2023; Published on 08/17/2023

ABSTRACT: This study was conducted to determine the effects of earthworm breeding O. cyanieum for 7, 14,

21, and 28 days in soil treated with concentrations of 800, 1000, 1500, and 2000 mg kg-1 of balanced NPK

compound fertilizer and balanced NPK nano-compound fertilizer at concentrations of 30, 60, 120, and 240 mg

kg-1, in addition to the control treatment. It was found that the weight and relative growth rate of earthworms

decreased with increasing concentrations of fertilizers. The highest significant decrease in weight (0.257 g) and

relative growth rate (40.34%) was observed at a concentration of 1500 mg kg-1 of balanced NPK compound

fertilizer. Meanwhile, the highest decrease in worm weight (0.225 g) and relative growth rate (86.31%) was

observed at a concentration of 240 mg kg-1 of balanced NPK nano-compound fertilizer. Fertilizers also affected

the protein content in the earthworms, which differed significantly from the control treatment for both

fertilizers. In addition, the fertilizers affect the histological structure of the body, with clear disruption in the

epithelium of the body, layers of circular and longitudinal muscle, the lining of the intestine, especially the

typhlosole.

Keywords: chemical fertilizer; nano fertilizer; earthworm vitality.

Efeito do fertilizante npk balanceado e nanocomposto na vitalidade da minhoca

octolasion cyanieum e seu efeito na composição histológica do meio do corpo

RESUMO: Este estudo foi conduzido para determinar os efeitos da criação de minhocas O. cyanieum por 7, 14,

21 e 28 dias em solo tratado com concentrações de 800, 1000, 1500 e 2000 mg kg-1 de solo de fertilizante

composto NPK balanceado (20-20-20) e fertilizante nanocomposto NPK balanceado (20-20-20) nas

concentrações de 30, 60, 120 e 240 mg kg-1 de solo, além do tratamento testemunha. O estudo mostrou que os

fertilizantes químicos tiveram impacto sobre a vitalidade dessas minhocas, pois o peso e a taxa de crescimento

relativo das minhocas diminuíram com o aumento das concentrações de fertilizantes. A maior queda

significativa no peso (0,257 g) e na taxa de crescimento relativo (40,34%) foi observada na concentração de

1500 mg kg-1 de solo do fertilizante composto NPK balanceado. Já a maior redução no peso (0,225 g) e na taxa

de crescimento relativo (86,31%) da minhoca foi observada na concentração de 240 mg kg-1 de solo do

fertilizante nanocomposto NPK balanceado. Além disso, os tratamentos acima tiveram impacto no teor de

proteína dos corpos das minhocas, que diferiu significativamente do tratamento controle, para ambos os tipos

de fertilizantes. O estudo mostrou o impacto do fertilizante nas estruturas dos tecidos das minhocas, com clara

ruptura na camada da pele e nas camadas musculares circulares e longitudinais abaixo dela, bem como um claro

efeito no revestimento intestinal, especialmente o tiflosole.

Palavras-chave: fertilizante químico; nano fertilizante; vitalidade da minhoca.

1. INTRODUCTION

Earthworms are invertebrates and an essential part of the

Earth's ecosystem since the pre-Cambrian era, which is about

600 million years ago. They belong to the phylum Annelida,

subclass Oligochaeta (PIEARCE et al., 1990). these

organisms act as natural plows for the soil and produce

vermicompost (Antunes et al, 2021), which is a highly

valuable material for meeting the nutritional needs of crops

(GHOSH, 2018; ASLAM et al., 2021).

Earthworms are considered one of the most important

large living organisms in the soil for maintaining and

controlling soil fertility. They play a vital role in renewing the

soil, and can only be found in soil with specific characteristics

such as texture, sufficient moisture, appropriate acidity levels,

and specific nutrients (KALE; KARMEGAM, 2010).

Environmental degradation resulting from human activities

can directly affect the quality of soil, and pose a threat to the

biodiversity of living organisms (MASIN et al., 2020).

According to some researchers, soil organisms that respond

to changes in the environment are considered to be of great

importance in maintaining soil quality, where non-vertebrate

animals form the highest biomass in the soil (MIGLANI;

BISHT, 2019; LUAN et al., 2020). Some researchers estimate

that earthworms may contribute to about 40-90% of the large

living biomass of soil organisms (AL-MALIKI et al., 2021;

HUANG et al.,2021). And since earthworms have the ability

Balanced and nano-composite NPK fertilizer on the vitality and histological composition …

Nativa, Sinop, v. 11, n. 2, p. 241-250, 2023.

242

to live in a variety of soil ecosystems, as well as their relatively

large size and sensitivity to many pollutants, and their ease of

cultivation due to their fast growth and short lifespan, they

play an important role in maintaining soil quality

(PODOLAK et al., 2020).

Earthworms can also be used for several generations to

assess the potential effects of persistent pollutants over

generations, as well as to study their ability to recover from

or tolerate the studied compounds (SCHNUG et al., 2013;

ŽALTAUSKAITĖ et al., 2020).

As increasing crop production largely depends on the

type of fertilizers used, different types of agricultural

chemicals such as chemical fertilizers are currently being used

excessively, which negatively affects the soil, as well as soil

plants and animals (MEENA et al., 2016; SENA et al., 2021).

Negative effects include the loss of topsoil due to pollution

from fertilizers, herbicides and pesticides, as well as the

bioaccumulation of chemicals in food chains and webs, The

use of excessive agricultural chemicals can have a negative

impact on soil ecosystems by weakening the physical,

chemical, and biological components of the soil, particularly

beneficial non-target microorganisms and earthworms

(DHANANJAYAN et al., 2020; GUNAWARDENA, 2022),

Fertilizers also increase the concentration of nitrates in the

water and this has a negative effect (AL-HUSSEIN et al.,

2023)

This study was conducted to determine the effect of

balanced NPK compound fertilizer and balanced NPK nano

compound fertilizer on the vitality of the earthworm

Octolasion cyanieum, as well as the effect on the tissue

composition of the skin and the middle part of the intestine.

2. MATERIAL AND METHODS

2.1. Collecting earthworm samples:

This study was conducted at the University of Mosul,

College of Education for Pure Sciences. Earthworms were

collected in late August 2022 from home gardens in the

Nimrud district south of Mosul. The samples were collected

by digging the soil to a depth of 15 to 30 cm or more and

were preserved and bred in plastic boxes with dimensions of

(55 * 35 * 28 cm) (length * width * height). The boxes

contained a piece of cloth placed at the bottom of the box

and extending to its sides. Bird droppings with soil were also

placed in these boxes for the purpose of feeding the worms

during their rearing period according to the method (GARG

et al., 2005; GARCZYŃSKA, 2020).

The soil was constantly moistened to keep the worms

alive and maintain their viability for use in laboratory

experiments. They were classified at the Research Center,

Natural History Museum/University of Baghdad,

Department of Insects and Invertebrates by Dr. Nabres Falih

Jijan Al-Khafaji. The breed is: Octolasion cyanieum (Savigny,

1826) (Crassiclitellata, Lumbricidae)

2.2. Fertilizers used in the study and preparation of

biological effects experiments on earthworms:

A completely randomized block design with three

replicates and five treatments per fertilizer, including one

control treatment and four other treatments, was used. The

current study used two types of fertilizers: inorganic chemical

fertilizer (balanced compound fertilizer NPK 20-20-20),

which was obtained from local markets in Mosul, Iraq, and

the following selected concentrations (800, 1000, 1500, and

2000 mg kg-1). Also, a nano-fertilizer (the balanced

compound nano-fertilizer 20-20-20-NPK), which was

obtained from the Green Fertilizer Company, and the

selected concentrations (30, 60, 120, and 240 mg kg-1) were

used. The concentrations used in the study were estimated

based on the recommended amounts per fertilizer kilogram/

Dunam, as well as selecting concentrations higher than the

recommended amounts. As well as, the necessary fertilizer

ratio for one treatment was determined by calculating the

amount of soil in kilograms at a depth of 15 centimeters per

Dunam, which was estimated at 500,000 kilograms per

Dunam. By performing the ratio and proportion operation

for each selected concentration of fertilizers, the necessary

amount of fertilizer in grams per kilogram of soil was

determined. Several bioassays were conducted to determine

the effect of fertilizers on the vitality of earthworms.

Plastic containers with a capacity of one kilogram were

taken and filled with 500 grams of soil that had been

previously sterilized at 90 degrees Celsius in an electric oven

for five hours. The purpose of sterilization was to ensure that

the soil was free of worm eggs or other organisms that could

interfere with the experiment's results. The soil's pH was 7.8,

and its moisture content was 17%. Before treating the

earthworms with fertilizer, they were placed in cups

containing distilled water for over two hours to clean their

digestive tract of soil and expel their waste. The worms'

weights were then measured using a sensitive electronic scale,

with 5 worms per repeater, ensuring they were approximately

the same size and age.

They were then placed in plastic containers containing

soil treated with fertilizers, which had been mixed well. Cattle

manure was used as food for the earthworms during the

experiment period, added every week after weighing the

worms. The plastic containers were then covered with a piece

of transparent cloth to prevent the worms from escaping

while being monitored for (7, 14, 21, and 28 days).

2.3. The effect of fertilizer concentrations on the weights

of earthworms:

Adult worms (with a clitellum) were selected, which were

similar in weight and age, and their weights were calculated

every week using a sensitive electronic scale, and the

difference in their weights was noted compared to the control

group.

2.4. The effect of fertilizers on the relative growth rate of

earthworms:

After measuring the weights of the worms before and after

treatment with the aforementioned fertilizers to determine

the extent of the effect of these fertilizers on the growth rate

of the worms, their growth rate was calculated according to

the method of Sogbesan; Ugwumba (2006) using the

following equation:

Relative growth rate (RGR) = weight after treatment ÷

weight before treatment × 100.

2.5. Estimation of protein content in the bodies of

earthworms:

The amount of protein in the bodies of earthworms

treated with fertilizers was estimated using the method of

(Lowry et al., 1951) and was modified by Schachterle; Pollack

(1973).

Mohammed & Mohammed

Nativa, Sinop, v. 11, n. 2, p. 241-250, 2023.

243

2.6. Histological tests:

For the purpose of determining the effect of the

fertilizers used in the experiments on the tissue structure of

some areas in the bodies of the earthworms treated with

fertilizers, histological tests were performed on these areas.

The samples were stained using the hematoxylin-eosin and

Delafield's hematoxylin stains based on the method of Luna

(1968) and Culling et al. (1985).

2.7. Examination and microscopic imaging of tissue

sections:

The tissue sections were examined using a compound

light microscope to observe the tissue effects resulting from

fertilizer treatment. After examination, the sections were

imaged using a microscope equipped with a digital camera.

The lens magnification power was calculated using the ratio

of the objective lens magnification to the eyepiece lens

magnification to determine the magnification power of the

tissue dimensions in the section.

2.8. Statistical Analysis:

The results were statistically analyzed using the SAS

program on a computer with a completely randomized

design. A factorial C.R.D. experiment was conducted, and

the differences between the means were selected using a

multiple range test (Duncan's test) at a 5% probability level

(L-ZUBAIDY; AL-FALAHY, 2016).

3. RESULTS

3.1. The effect of balanced NPK fertilizer

The results in Table (1) show that treating soil with

concentrations of 800, 1000, 1500, and 2000 mg kg-1 of

balanced NPK fertilizer for cultivation periods of 7, 14, 21,

and 28 days had a significant effect on the weight rate of O.

cyanieum earthworms. We find from the table that the

different concentrations had a clear effect, as the weight rate

significantly decreased, reaching 0.458 and 0.545 g at

concentrations of 1500 and 2000 mg kg-1, respectively. Both

concentrations were significantly different from the weight

rate of the control treatment, which was 0.832 g.

As Table 1 shows, the weight rate was also affected by the

duration of the earthworms' stay in the environment treated

with NPK fertilizer. The weight rate decreased with the

increase of the time period, with the weight being 0.662,

0.660, 0.625, and 0.570 g at time periods of 7, 14, 21, and 28

days, respectively.

As for the effect of the interaction between concentration

and time period, we notice that all the earthworms died at a

concentration of 2000 mg kg-1 starting from the time period

of 14 days of treatment. As we can see from the table, in the

first time period of treatment, which was 7 days, all

concentrations showed a significant difference with the

comparison group, which had a weight of 0.752 g. The lowest

weight of the earthworms was 0.545 g at a concentration of

2000 mg kg-1, and 0.645, 0.664, and 0.708 g at concentrations

of 1500, 1000, and 800 mg kg-1, respectively, and for the same

time period.

In the time periods of 14 and 21 days, there was also a

significant decrease in the weight of the earthworms, as

shown in the table, compared to the comparison group,

which had weights of 0.804 and 0.864 g in the mentioned

time periods, respectively. The highest weight decrease was

recorded in the 28-day period, which was 0.257 g at a

concentration of 1500 mg kg-1, and 0.571 and 0.544 g at

concentrations of 800 and 1000 mg kg-1, respectively, no

significant difference between them. The weight of the

comparison group was 0.911 g for the same time period,

which was significantly different.

Table 1. The average weight (g) of the earthworm O. cyanieum raised in soil treated with different concentrations of NPK compound fertilizer

for several time periods.

Tabela 1. Peso médio (g) da minhoca O. cyanieum criada em solo tratado com diferentes concentrações de fertilizante composto NPK por

vários períodos de tempo.

Concentration (mg kg-1) Exposure period (days)

7 14 21 28 Concentration effect rate

800 0.708 e 0.672 f 0.644 gh * 0.571 j 0.648 b

1000 0.664 fg 0.621 hi 0.607 i 0.544 j 0.609 c

1500 0.645 gh 0.545 j 0.386 k 0.257 l 0.458 d

2000 0.545 j … … ** … 0.545 e

control 0.752 d 0.804 c 0.864 b 0.911 a 0.832 a

Rate (time period effect) 0.662 a 0.660 ab 0.625 b 0.570c

*Numbers that carry different letters have different meanings according to the Duncan multiple range test at a significance level of 0.05%. ** (.....) means the

death of all worms during the recorded period.

The results in Table 2 show that treating the soil with

concentrations of 800, 1000, 1500, and 2000 mg kg-1 of NPK

compound fertilizer for the cultivation periods of 7, 14, 21,

and 28 days had a significant effect on the relative growth

rate of the earthworm O. cyanieum. From the table, it can be

seen that the different concentrations have a clear effect, with

a significant decrease in the relative growth rate to 61.34%

and 73.35% at concentrations of 1500 and 2000 mg kg-1,

respectively, and both concentrations differed significantly

from the relative growth rate in the control treatment, which

reached 113.60%.

As the table shows, the relative growth rate was also

affected by the treatment period, with the lowest growth rate

being 77.41% and 84.73% at the treatment periods of 28 and

21 days, respectively, meaning that the relative growth rate

decreased with an increase in the exposure period. As for the

effect of the interaction between concentration and

treatment time, we notice that all worms died at a

concentration of 2000 mg kg-1 and for treatment periods of

14, 21, and 28 days, respectively. The table also shows a

significant difference in the relative growth rate during the

first (7 days) of cultivation, as the lowest relative growth rate

was 73.35% at a concentration of 2000 mg kg-1. Similarly, the

relative growth rates at the other concentrations showed a

clear decrease compared to the comparison group which had

a relative growth rate of 102.59% for the same time period.

Balanced and nano-composite NPK fertilizer on the vitality and histological composition …

Nativa, Sinop, v. 11, n. 2, p. 241-250, 2023.

244

Additionally, we observe a significant decrease in the relative

growth rate of the worms during the second treatment period

of 14 days and the third period of 21 days. The maximum

relative growth rate during the second period was 91.42% at

a concentration of 800 mg kg-1, while the relative growth rate

for the comparison group was 109.68% during this period.

Furthermore, the maximum relative growth rate during the

third period of 21 days was 87.61% at a concentration of 800

mg kg-1, while the comparison group had a relative growth

rate of 117.87% during the same period. As for the fourth

time period of 28 days, we observe the highest decrease in

the relative growth rate of 34.40% at a concentration of 1500

mg kg-1, and 73.31% and 77.68% at concentrations of 1000

mg kg-1 and 800 mg kg-1, respectively. All of the above results

were significantly different from the comparison group,

which had a relative growth rate of 124.28% during the same

time period (28 days).

Table 2. Relative growth rate (%) of earthworm O. cyanieum raised in soil treated with different concentrations of NPK fertilizer for various

periods of time.

Tabela 2. Taxa de crescimento relativo (%) da minhoca O. cyanieum criada em solo tratado com diferentes concentrações de fertilizante

composto NPK por vários períodos de tempo.

Concentration (mg kg-1) Exposure period (days)

7 14 21 28 Rate (concentration effect)

800

96.32 e

91.42 f

87.61 fh

* 77.68 jk

88.17 b

1000

89.48 fg

83.69 hi

81.80 ij

73.31 k

82.08 c

1500

86.34 gi

72.95 k

51.67 l

34.40 m

61.34 d

2000

73.35 k

…

... **

…

73.35 e

control

102.59 d

109.68 c

117.87 b

124.28 a

113.60 a

Rate (time period effect) 89.61 a 89.43 ab 84.73 b 77.41 c

*Numbers that carry different letters have different meanings according to the Duncan multiple range test at a significance level of 0.05%. ** (.....) means

the death of all worms during the recorded period.

The results in Table (3) show the treatment of soil with

concentrations of 800, 1000, 1500, and 2000 mg kg-1 of

balanced NPK compound fertilizer and its effect on the

protein content in the body of earthworms O. cyanieum after

28 days, except for the concentration of 2000 mg/ kg-1

(protein was measured after seven days). There was a

significant difference in the amount of protein recorded,

which was 0.142, 0.166, and 0.216 mg g at concentrations of

800, 1000, and 1500 mg kg-1, respectively. And all of these

concentrations showed a significant decrease compared to

the control group (0.250 mg g), The protein content of the

worm increased, reaching 0.233 mg g at a concentration of

2000 mg kg-1. Despite its increase, there was no significant

difference with the control group.

Table 3. Protein content (mg g) in the body of earthworms O.

cyanieum raised in soil treated with different concentrations of NPK

compound fertilizer and for different periods of time.

Tabela 3. Teor de proteína (mg g) no corpo de minhocas O. cyanieum

criadas em solo tratado com diferentes concentrações de fertilizante

composto NPK e por diferentes períodos de tempo.

control

800

1000

1500

2000

The amount of

protein

0.25

0.14d

0.166c

0.21b

*0.233ab

*Numbers that carry different letters have different meanings according to

the Duncan multiple range test at a significance level of 0.05%

The results in Table (4) indicate that soil treatment with

concentrations of 30, 60, 120, and 240 mg kg-1 of balanced

nano NPK fertilizer for cultivation periods of 7, 14, 21, and

28 days had a significant effect on the weight of earthworms

O. cyanieum. From Table 4, it is clear that different

concentrations had a distinct effect, as the weight

significantly decreased to 0.683 and 0.451 g at concentrations

of 120 and 240 mg kg-1, respectively. Both treatments were

statistically different from the weight average. The weight at

the comparison treatment was 0.833 g.

The table also shows that the weight rate was also affected

by the length of time the worm stayed in the soil treated with

balanced nano NPK fertilizer. The weight rate decreased with

an increase in the time period, and the weight was 0.749,

0.714, 0.663, and 0.592 g for the time periods of 7, 14, 21,

and 28 days, respectively. There was a significant difference

between these treatments.

As for the effect of the interaction between concentration

and time period, we notice from the table that in the first time

period of 7 days, all concentrations recorded a significant

difference from the comparison group, which had a weight

of 0.782 g, except for the weight at a concentration of 60 mg

kg-1, which did not record a significant difference. We note

that the weight at a concentration of 30 mg kg-1 was higher

than that in the comparison group for the same period. In

the 14- and 21-day periods, there was also a significant

decrease in the weights of the worms, as shown in Table 4.

The lowest weight was 0.530 and 0.417 g at a concentration

of 240 mg kg-1, compared to the comparison group, which

weighed 0.812 and 0.844 g in the respective periods. As for

the 28-day period, we note that there was no significant

difference in weights between the concentrations of 30 and

60 mg kg-1, but both recorded a significant difference from

the comparison group, which weighed 0.894 g in the same

time period.

3.2. The effect of balanced NPK nanocomposite

fertilizer

The results in Table 5 show that treating the soil with

concentrations of 30, 60, 120, and 240 mg kg-1 of balanced

NPK nanocomposite fertilizer for cultivation periods of 7,

14, 21, and 28 days had a significant effect on the relative

growth rate of O. cyanieum earthworms. We can see from the

table that the different concentrations had a clear significant

effect, as the relative growth rate significantly decreased. The

growth rate percentage was 103.06% at a concentration of 30

mg kg-1, while it was 63.87% at a concentration of 240 mg kg-

1. Table 5 also shows that the relative growth rate was

significantly affected by the treatment period. The lowest

growth rate was 94.55% and 84.46% for treatment periods of

21 and 28 days, respectively, indicating that the relative

growth rate decreased with an increase in exposure time.

Mohammed & Mohammed

Nativa, Sinop, v. 11, n. 2, p. 241-250, 2023.

245

As for the effect of the interaction between concentration

and treatment period, we can see from the table that there

was a significant difference in the relative growth rate in the

first period of cultivation, which was (7 days), All

concentrations showed a significant difference in the growth

rate compared to the control group, which was 111.23%. We

also notice that the concentrations 30 and 60 mg/ kg-1 did

not significantly differ in the relative growth rate. Similarly,

we observe a significant decrease in the relative growth rate

during the second period of cultivation, which was (14 days).

The growth rate ranged from 109.42% to 75.07% in

concentrations of 30 and 240 mg/ kg-1, respectively,

compared to the control group of 115.50%. During the third

period of cultivation, which was 21 days, there was a

significant decrease in the relative growth rate of the

earthworms. The maximum growth rate during this period

was 99.14% at a concentration of 30 mg/ kg-1, while the

control group had a growth rate of 120.05%. During the

fourth time period of (28 days), we observed a significant

difference between the concentrations and the comparison

group, was the highest decrease in relative growth rate being

31.86% at a concentration of 240 mg/ kg-1, while the

comparison group recorded a growth rate of 127.16% during

the same time period.

Table 4. The average weight (g) of O.cyanieum earthworms raised in soil treated with different concentrations of NPK nanocomposite

fertilizer for several periods of time.

Tabela 4. Peso médio (g) de minhocas O. cyanieum criadas em solo tratado com diferentes concentrações de fertilizante nanocompósito NPK

por vários períodos de tempo.

Concentration (mg kg-1) Exposure period (days)

7 14 21 28 Rate (concentration effect)

0.788 bd

0.766 df

0.690 gh

* 0.636 i

0.720 b

0.781 ce

0.734 dg

0.709 fg

0.626 i

0.712 c

120

0.764 df

0.730 eg

0.656 hi

0.582 j

0.683 c

240

0.632 i

0.530 j

0.417 k

0.225 l

0.451 d

control

0.782 ce

0.812 bc

0.844 b

0.894 a

0.833 a

Rate (time period effect)

0.749 a

0.714 b

0.663 c

0.592 d

*Numbers that carry different letters have different meanings according to the Duncan multiple range test at a significance level of 0.05%.

Table 5. Relative growth rate (%) of O.cyanieum earthworms raised in soil treated with different concentrations of NPK nanocomposite

fertilizer for several periods of time.

Tabela 4. Taxa de crescimento relativo (%) de minhocas O. cyanieum criadas em solo tratado com diferentes concentrações de fertilizante

NPK nanocomposto por vários períodos de tempo.

Concentration (mg kg-1) Exposure period (days)

7 14 21 28 Rate (concentration effect)

112.57 dc

109.42

99.14 hi

* 91.14

103.06 b

112.21 dc

105.45

101.86

89.94 jk

102.36 bc

120

107.90 dh

103.10

92.65 ij

82.20 k

96.46 c

240

89.51 jk

75.07 l

59.06 m

31.86 n

63.87 d

control

111.23 de

115.50

120.05

127.16 a

118.48 a

Rate (time period effect)

106.68 a

101.70

94.55 c

84.46 d

*Numbers that carry different letters have different meanings according to the Duncan multiple range test at a significance level of 0.05%.

The results in Table (6) show the effect of soil treatment

with concentrations of 30, 60, 120, and 240 mg kg-1 of nano

NPK compound fertilizer on the protein content in the body

of O. cyanieum earthworms after 28 days. We observe a

significant decrease in protein content, with the lowest

protein percentage of 0.043 mg g occurring at a

concentration of 30 mg kg-1. The protein content was 0.075,

0.116, and 0.193 mg g at concentrations of 60, 120, and 240

mg kg-1, respectively, and all showed a significant difference

from the control group (0.250 mg g).

Table 6. Protein content (mg g) in the body of O. cyanieum

earthworms raised in soil treated with different concentrations of

NPK nano compound fertilizer for various time periods.

Tabela 3. Teor de proteína (mg g) no corpo de minhocas O. cyanieum

criadas em solo tratado diferentes concentrações de fertilizante

NPK nanocomposto e por diferentes períodos de tempo.

control

120

240

The amount

of protein

0.25a

0.043e

0.075d

0.116c

*0.193b

*Numbers that carry different letters have different meanings according to

the Duncan multiple range test at a significance level of 0.05%

4. DISCUSSION

4.1. Effect of balanced NPK compound fertilizer and

balanced NPK nano compound fertilizer on the weight

and relative growth rate of earthworms

Table 1 shows that the weight of earthworms decreased

as the concentration and duration of exposure to balanced

NPK fertilizer increased over periods of 7, 14, 21, and 28

days. Similarly, the relative growth rate of earthworms

decreased as the concentration and duration of exposure

increased, as shown in Table 2. These results demonstrate

that treating soil with NPK compound fertilizer

concentrations of 800, 1000, 1500, and 2000 mg kg-1 for

cultivation periods of 7, 14, 21, and 28 days resulted in a

decrease in the relative growth rate of earthworms. This

decrease in relative growth rate was more pronounced with

increased concentration and duration of exposure.

According to the results shown in Table 4, the treatment

of soil with concentrations of 30, 60, 120, and 240 mg kg-1 of

balanced nanoscale NPK composite fertilizer, and for the

cultivation periods of 7, 14, 21, and 28 days, resulted in an

increase in worm weight of 0.788 g in the first period of 7

days at a concentration of 30 mg kg-1 compared to the control

Balanced and nano-composite NPK fertilizer on the vitality and histological composition …

Nativa, Sinop, v. 11, n. 2, p. 241-250, 2023.

246

group, as previously mentioned in the results. Also, the

relative growth rate for the same fertilizer and the same

cultivation period of 7 days increased at concentrations of 30

and 60 mg kg-1, reaching 112.57% and 112.21%, respectively.

This is similar to the results obtained by RAI et al. (2014) and

is consistent with Long et al. (2017) when treating worms

with urea at concentrations of 50, 100 and 150 mg kg-1. There

may be two explanations for this: first, the bodies of the

earthworms exposed to the fertilizers swelled, leading to an

increase in weight. The other explanation is that the increase

in the weight of the earthworms was a healthy increase (RAI

et al., 2014).

Despite what was mentioned, the weight of the worms

for the above concentrations and other concentrations of 120

and 240 mg kg-1 decreased with an increase in the time period.

Also, the results in Table 5 showed that the relative growth

rate of worms treated with balanced nano NPK compound

fertilizer for the same concentrations and for a period of 28

days decreased with an increase in the concentrations and

exposure time, except for what was mentioned above.

The reason for the decrease in the weight and relative

growth rates of the worms may be due to the physiological

stress that the worms were exposed to as a result of the

changes that occurred in the environment due to the addition

of fertilizers. It has been found that adding urea fertilizer to

the soil leads to changes in the soil chemistry, such as changes

in the soil pH and differences in ammonia levels. It has also

been shown that these changes impact soil organisms,

including earthworms (STALEY et al., 2018). Our results are

consistent with the study by ZHANG et al. (2022) in their

study on the effect of polyethylene nanoparticles (PE MPs)

and zinc oxide nanoparticles (ZnO NPs) on the earthworm

E. fetida. The application of these materials led to a decrease

in worm weights. Our results also match Yan et al. (2021) in

their study on the combined effect of neonicotinoid

pesticides and heavy metals on earthworms, which had a

negative impact on growth rate. Our current study's results

are similar to QIAO et al. (2019) in their study on the

earthworm E. fetida. Treating earthworms with five

concentrations of the insecticide cyantraniliprole for 28 days

resulted in a significant effect on the growth rate of the

worms.

4.2. Effect of balanced NPK compound fertilizer and

NPK nano compound fertilizer on the protein content

in the body of earthworms

From the results in Table 3, treating the soil with

concentrations of 800, 1000, 1500, and 2000 mg kg-1 of

balanced NPK compound fertilizer, and the results in Table

6, treating the soil with concentrations of 30, 60, 120, and 240

mg kg-1 of balanced NPK nanocomposite fertilizer, showed

that the fertilizers affected the protein content in the body of

earthworms. Similarly, our findings matched those of

SAMAL et al. (2017) in their study of the effect of urea,

phosphate gypsum, and paper mill sludge on the earthworms

Drawida willsi and Lampito mauritii, as the protein content in

the bodies of the earthworms decreased significantly with the

decrease in the concentration of urea and phosphate gypsum.

Our results were also consistent with those of MOSLEH et

al. (2003), as exposure of the earthworms E. fetida to

pesticides caused a significant decrease in their protein

content. This was attributed to a lower food intake in the

earthworms exposed to pesticides compared to the control

group (RIBERA et al., 2001). We hypothesize that the same

applies to the chemical fertilizers used in our current study.

4.3. Effect of balanced compound NPK fertilizer and

balanced NPK nano compound fertilizer on the tissue

composition of earthworm

O. cyanieum

Adeel et al. (2021) stated that any injury to the external

skin and intestines may have harmful effects on the health of

earthworms, which delays the normal performance of these

tissues and may lead to earthworm mortality. Additionally,

tissue injury affects the vital energy of earthworms, which

ultimately leads to a disturbance in the overall energy budget,

leaving less energy for physiological processes such as growth

and reproduction.

Therefore, our results matched those of SAMAL et al.

(2017) in their study, where damage occurred to the skin and

surface tissues, separation between longitudinal and circular

muscles, and decay occurred. Also, the connective tissues

were affected when worms were treated with urea, phosphate

gypsum, and paper mill sludge. Our results also matched

those of WANG et al. (2020) in their study where tissue

damage occurred in the body wall, intestine, and seminal

vesicles of earthworms under a high level of exposure to

nickel. Our results also matched those of Lahive et al. (2014)

in their study on the effect of cerium oxide nanoparticles

(NPs) on the earthworms E. fetida, the exposure led to

significant damage to the skin and surface layer. Our study

also matched that of Adeel et al. (2021), where the application

of rare earth metals, including nano and bulk lanthanum

(La2O3) and yttrium oxides (Yb2O3), on earthworms caused

radical changes in the epithelium layer of the digestive system

and typhlosol. The epidermis of the Earthworm treated with

200 mg kg-1 of nanominerals and ordinary metals has been

sucked and peeled. Applying the same substances with the

same concentration also led to edema in the layers of

longitudinal muscle and circular muscle inflammatory cells in

the intestinal tissue of the earthworm. Also, the occurrence

of necrosis in mucous cells, compared with the untreated

control group.

4.4. Figures The effect of balanced NPK compound

fertilizer and NPK nano compound fertilizer on the

tissue structure of

O. cyanieum

earthworms

To understand the mechanism of the effect of the

fertilizer used in this study, tissue sections were made of the

middle part of the earthworm body. Figures 1 and 2, show a

section of an untreated earthworm, illustrating the

composition of the body wall, which consists of the skin

layer, circular and longitudinal muscles, and the wall part of

the peritoneal membrane. The section also shows the middle

part of the intestine and the blind area of the typhlosole.

Figure 3 shows the effect of breeding earthworms for 14

days in soil treated with a concentration of 1500 mg kg-1 of

the balanced NPK compound fertilizer. This led to an

increase in the thickness of the circular and longitudinal

muscle layers, as well as the separation and detachment of

fibers from these muscle layers. The treatment also resulted

in the separation of the cuticle layer from the skin, causing

congestion of blood vessels within the muscle layers and

necrosis of skin cells.

Similarly, breeding these worms for 21 days in soil treated

with the same previous concentration of fertilizer led to an

inflammation in the intestinal lining and a clear separation of

Mohammed & Mohammed

Nativa, Sinop, v. 11, n. 2, p. 241-250, 2023.

247

the peritoneal membrane from the muscle layer, with edema

occurring in the connective tissue of the typhlosole. Folds

were also present in the mucous membrane lining the

intestines and typhlosole (Figure 4).

As shown in Figure 5, after 28 days of breeding with the

same concentration of fertilizer, there was inflammation in

the circular muscle layer and a separation between the fibers

of the longitudinal muscle layer, as well as a separation of the

peritoneal membrane from the muscle layer. There were also

bulges in the skin layer and separation of the cuticle layer.

As shown in Figure 6, breeding O. cyanieum earthworms

for seven days with a concentration of 2000 mg kg-1 of

balanced NPK compound fertilizer led to a separation of the

cuticle layer, with a clear increase in inflammation of the skin

layer cells and a separation of the fibers of the circular muscle

layer, as well as inflammation in the longitudinal muscle layer.

Figure 7 shows that the balanced nano NPK compound

fertilizer also had an effect on the histological structure of the

earthworms' body medium. Breeding earthworms for 28 days

in soil treated with a concentration of 60 mg kg-1 of the

fertilizer led to a separation of the cuticle layer from the skin

layer, with an increase in the pigmentation of the connective

tissue under the circular muscle layer, and a separation in the

longitudinal muscle layer. In addition, there was an increase

in the thickness of the skin layer in some areas.

Figure 1. A cross-sectional view of the control group of the

earthworm midgut, showing (1) skin layer, (2) circular muscles, (3)

longitudinal muscle layer, (4) peritoneal membrane. The image is

colored with E and H stains and has a magnification of 40x.

Figura 1. Corte transversal do grupo controle do intestino médio da

minhoca, mostrando: (1) camada da pele, (2) músculos circulares,

(3) camada muscular longitudinal e (4) membrana peritoneal. A

imagem é colorida com manchas E e H e tem uma ampliação de

40x.

Figure 2. Cross-sectional view of the midgut of an earthworm from

the control group. The image shows: (1) blind channel, (2) hepatic

cells located within the blind channel's invagination, (3) lining of the

digestive channel.

Figura 2. Corte transversal do intestino médio de uma minhoca do

grupo de controle. A imagem mostra: (1) canal cego, (2) células

hepáticas localizadas na invaginação do canal cego, (3) revestimento

do canal digestivo.

Figue 3. Cross-sectional view of the midgut of an earthworm that

was raised for 14 days in soil treated with a balanced NPK

compound fertilizer at a concentration of 1500 mg kg-1. The image

shows: (1) an increase in the thickness of the circular muscle layer,

(2) an increase in the thickness of the longitudinal muscle layer and

the occurrence of gaps and separations between its fibers, (3)

separation of the cuticle layer, (4) congestion of blood vessels within

the circular muscle layer, and (5) erosion of skin cells.

Figura 3. Corte transversal do intestino médio de uma minhoca que

foi criada por 14 dias em solo tratado com fertilizante composto

NPK balanceado na concentração de 1500 mg kg-1. A imagem

mostra: (1) aumento da espessura da camada muscular circular, (2)

aumento da espessura da camada muscular longitudinal e ocorrência

de lacunas e separações entre suas fibras, (3) separação da camada

de cutícula, (4) congestão dos vasos sanguíneos dentro da camada

muscular circular e (5) erosão das células da pele.

Figure 4. Cross-sectional view of the midgut of an earthworm that

was raised for 21 days in soil treated with a balanced NPK

compound fertilizer at a concentration of 1500 mg kg-1. The image

shows: (1) inflammation in the intestinal lining, (2) separation of the

peritoneal membrane from the longitudinal muscle layer, (3)

congestion in blood vessels, (4) edema within the connective tissue

of the blind channel cells, and (5) the appearance of folds in the

mucous membrane that lines the intestine and blind channel.

Figura 4. Visão transversal do intestino médio de uma minhoca que

foi criada por 21 dias em solo tratado com fertilizante composto

NPK balanceado na concentração de 1500 mg kg-1. A imagem

mostra: (1) inflamação no revestimento intestinal, (2) separação da

membrana peritoneal da camada muscular longitudinal, (3)

congestão nos vasos sanguíneos, (4) edema dentro do tecido

conjuntivo das células do canal cego e ( 5) o aparecimento de dobras

na membrana mucosa que reveste o intestino e o canal cego.

1 2

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Nativa, Sinop, v. 11, n. 2, p. 241-250, 2023.

248

Figure 5. Cross-sectional view of the midgut of an earthworm that

was raised for 28 days in soil treated with a balanced NPK

compound fertilizer at a concentration of 1500 mg kg-1. The image

shows: (1) inflammation in the circular muscle layer, (2) separation

between the fibers of the longitudinal muscle layer, (3) separation of

the peritoneal membrane from the muscle layer, (4) separation of

the cuticle layer, and (5) the appearance of bulges in the skin layer.

Figura 5. Visão transversal do intestino médio de uma minhoca que

foi criada por 28 dias em solo tratado com fertilizante composto

NPK balanceado na concentração de 1500 mg kg-1. A imagem

mostra: (1) inflamação na camada muscular circular, (2) separação

entre as fibras da camada muscular longitudinal, (3) separação da

membrana peritoneal da camada muscular, (4) separação da camada

de cutícula e (5) o aparecimento de protuberâncias na camada da

pele.

Figure 6. Cross-sectional view of the midgut of an earthworm that

was raised for seven days in soil treated with a balanced NPK

compound fertilizer at a concentration of 2000 mg kg-1. The image

shows: (1) separation of the skin layer from the circular muscle layer,

(2) clear inflammation in the skin layer cells, (3) separation in the

circular muscle layer, and (4) separation and inflammation in the

longitudinal muscle layer.

Figura 6. Visão transversal do intestino médio de uma minhoca que

foi criada por sete dias em solo tratado com fertilizante composto

NPK balanceado na concentração de 2.000 mg kg-1. A imagem

mostra: (1) separação da camada da pele da camada muscular

circular, (2) inflamação clara nas células da camada da pele, (3)

separação na camada muscular circular e (4) separação e inflamação

na camada muscular longitudinal.

Figure 8 shows that breeding earthworms for the same

duration as before but at a higher concentration caused

nibbling in the skin layer, as well as separation in some areas

of the circular muscles with edema. There was also

degradation and separation of the longitudinal muscle fiber

layer. The intestines were also affected, with finger-like

protrusions appearing in their lining, and separation from the

surrounding muscle layer.

Figure 7. Cross-sectional view of the midgut of an earthworm that

was raised for 28 days in soil treated with a balanced nanoscale NPK

compound fertilizer at a concentration of 60 mg kg-1. The image

shows: (1) separation of the cuticle layer, (2) an increase in

connective tissue pigmentation under the circular muscle layer, (3)

separation in the longitudinal muscle layer, and (4) an increase in the

thickness of the skin layer in some areas.

Figura 7. Visão transversal do intestino médio de uma minhoca que

foi criada por 28 dias em solo tratado com fertilizante composto

NPK em nanoescala balanceado na concentração de 60 mg kg-1. A

imagem mostra: (1) separação da camada da cutícula, (2) aumento

da pigmentação do tecido conjuntivo sob a camada muscular

circular, (3) separação da camada muscular longitudinal e (4)

aumento da espessura da camada da pele em algumas áreas.

Figure 8. Cross-sectional view of the midgut of an earthworm that

was raised for 28 days in soil treated with a balanced nanoscale NPK

compound fertilizer at a concentration of 240 mg kg-1. The image

shows: (1) presence of erosion in some areas of the skin layer, (2)

presence of separation in some areas of the circular muscle layer, (3)

degradation and separation of the fibers of the longitudinal muscle

layer, (4) appearance of finger-like protrusions in the lining of the

intestine of the blind channel, (5) separation of the intestinal lining

from the muscle layer, and (6) appearance of edema between the

fibers of the circular muscle layer.

Figura 8. Visão transversal do intestino médio de uma minhoca que

foi criada por 28 dias em solo tratado com fertilizante composto

NPK em nanoescala balanceado na concentração de 240 mg kg-1. A

imagem mostra: (1) presença de erosão em algumas áreas da camada

da pele, (2) presença de separação em algumas áreas da camada

muscular circular, (3) degradação e separação das fibras da camada

muscular longitudinal, (4) aparecimento de saliências semelhantes a

dedos no revestimento do intestino do canal cego, (5) separação do

revestimento intestinal da camada muscular e (6) aparecimento de

edema entre as fibras da camada muscular circular.

5. CONCLUSIONS

The study showed that the improper use of chemical

fertilizers causes soil pollution and has a clear impact on the

vitality of earthworms. These fertilizers (conventional NPK

Mohammed & Mohammed

Nativa, Sinop, v. 11, n. 2, p. 241-250, 2023.

249

and nano) had a significant effect on the relative growth,

weight, and protein content of earthworms, as well as on the

tissue structure of the skin, circular and longitudinal muscles,

and intestinal lining.

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Acknowledgments: The authors would like to express their sincere

thanks and appreciation to the presidency of the University of

Mosul and the College of Education for Pure Sciences at the

University of Mosul, for their support in presenting this research

paper in the best possible way

Author Contributions: DH.H.M. – data collection, writing and

draft writing; A.M.M. – methodology, statistical analysis,

proofreading; management and supervision. All authors read and

agreed to the published version of the manuscript.

Funding: Not applicable.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement (Ethics Committee of the area):

Not applicable.

Data Availability Statement (how the data can be made

available): Raw and analyzed data can be obtained by request to the

corresponding Author by e-mail.

Conflicts of Interest: The authors declare no conflict of interest.

Supporting entities had no role in the design of the study; in the

collection, analysis, or interpretation of data; in the writing of the

manuscript, or in the decision to publish the results