Introduction

Background of the Study

Most species of the genus Panaeolus are saprobes that decompose grass litter,
dung, or forest litter and most require microscopic examination for accurate
identification. (Gerhardt, E., 1996) Panaeolus antillarum (Fr.) Dennis is a common and
widely distributed small to medium sized edible grey mushroom which grows on dung or
manure of cattle and sometimes horses. The caps are 3 to 7 cm, bell-shaped to convex,
white to light gray or yellowish, thick, smooth, often with fine wrinkles and acquire a
silver white shiny color in age. Its gills are gray in young specimens, turning black as the
spores mature. Its stipe, which can grow from 4 to 22 cm long and .5 to 2 cm thick,
solid, are sometimes slightly larger at the base. (Gerhardt E., 1987)
Bioindicators are organisms or communities of organisms, which reactions are
observed representatively to evaluate a situation, giving clues for the condition of the
whole ecosystem. The bioindicator has particular requirements with regard to a known
set of physical or chemical variables such that changes in presence or absence,
numbers, morphology, physiology or behavior of that species indicate that the given
physical or chemical variables are outside their preferred limits. Specifically,
bioindicators are a species or group of species that readily reflects the abiotic or biotic
state of an environment, represents the impact of environmental change on a habitat,
community or ecosystem or is indicative of the diversity of a subset of taxa or the whole
diversity within an area. (A. Gerhardt, 1995)

Statement of the Problem

This study aimed to analyze the mycelial growth of Panaeolus Antillarum on
different heavy metals.
Specifically, this study seeks answers to the following questions:
1. Are there differences on the growth of Panaeolus Antillarum mycelia on different
heavy metals concentrations?
2. Are there differences on the average linear mycelial growth of Panaeolus
Antillarum on different heavy metals concentrations?
3. Are there differences on the inhibition of Panaeolus Antillarum on different heavy
metals concentrations?
4. Are there differences on the density of Panaeolus Antillarum on different heavy
metal concentrations?
Hypotheses:
1. There were no differences on the growth of mycelia in different heavy metal
concentrations.
2. There were no differences on the average linear mycelial growth of
Panaeolus antillarum in different heavy metal concentrations.
3. There were no differences on the inhibition of Panaeolus antillarum in
different heavy metal concentrations.
4. There were no differences on the density of Panaeolus Antillarum on different
heavy metal concentrations.

Significance of the Study

The study was conducted to help identify the conditions of different environments
or ecosystems where the mushroom grows. The size of the mushroom and the rate it
grows in an environment indicates a certain lack or abundance of an element of the soil
2

or medium. In this case, the heavy metal contamination in the soil or medium where the
mushroom grows is indicated. The mushroom growth relies on the condition of the
environment or media and according to its preferred limits. The degree of pollution and
heavy metal contamination in an ecosystem can be indicated by the mushroom and this
can be the basis for the steps to be done to clean the environment.
Scope and Limitations
This study was limited on the study of Panaeolus Antillarum as bioindicator in
Zinc Sulfate, Copper Sulfate and Lead Sulfate contaminated media.
The mycelia were cultured at Central Luzon State University, Munoz, Nueva Ecija
and were used as inoculums for each agar. The heavy metal concentrations used as
parameters for the study were: 1 ppm Zinc Sulfate (ZnSO4),10 ppm Zinc Sulfate
(ZnSO4), 100 ppm Zinc Sulfate (ZnSO4),1 ppm Copper Sulfate (CuSO4),10 ppm
Copper Sulfate (CuSO4), 100 ppm Copper Sulfate (CuSO4),1 ppm Lead Sulfate
(PbSO4), 10 ppm Lead Sulfate (PbSO4) and 100ppm Lead Sulfate (PbSO4).
This study was conducted from January to February 2012. The heavy metals
concentrations and inoculation were done at Central Luzon State University. And the
mycelia growth was measured and evaluated for its reaction to heavy metals.

Review of Related Literatures and Studies

Dabrowska et.al., (2012) used roe and red deer as bioindicators of heavy
metal contamination in Northwestern Poland. They examined the concentration of
3

heavy metals (Lead, Copper, Cadmium, Iron and Zinc) in the selected organs of roe and
red deer (liver and kidney). They found out that the concentration of Cadmium was
higher than that of lead. They were able to determined that the Cadmium and Lead
were present in that particular area
Fernandes et.al.,studied heavy metals in wild edible mushrooms under different
pollution conditions by x-ray fluorescence. They compared the metal uptake in Lepiota
procera,Boletus

badius,

Tricholoma

equestry, Lactarius

deliciosus,

Cantarelus

tubalformis and Cantarelus tubalformis , relative to sampling sites submitted to different

pollution conditions. They found out that mushrooms are suitable as bioindicator for
studying the environmental contamination by heavy metals.
According to Gerhadt A. (2011), boindicators are organisms which are use to
evaluate situations, giving clues for the condition of the whole ecosystem. They are
species that react to anthropogenical effects of the environment, whereas bioindicators
for natural environment changes and conditions are not much used. However, a
general, all ecompassing definition of biological indicator would be: a species or group
of species that readily reflects the abiotic or biotic state of an environment, represents
the impact of environmental change on a habitat, community or ecosystem or is
indicative of diversity of a subset of taxa or the whole diversity in the area.
According to Gerhardt E.(1987) most species of the genus Panaeolus
are saprobes that decompose grass litter, dung, or forest litter and most require
microscopic examination for accurate identification. (Gerhardt, E., 1996) Panaeolus
antillarum (Fr.) Dennis

is a common and widely distributed small to medium sized

edible grey mushroom which grows on dung or manure of cattle and sometimes horses.
4

The caps are 3 to 7 cm, bell-shaped to convex, white to light gray or yellowish, thick,
smooth, often with fine wrinkles and acquire a silver white shiny color in age. Its gills are
gray in young specimens, turning black as the spores mature. Its stipe, which can grow
from 4 to 22 cm long and .5 to 2 cm thick, solid, are sometimes slightly larger at the
base.
Gerhardt A. (1995) wrote that bioindicators are organisms or communities of
organisms, which reactions are observed representatively to evaluate a situation, giving
clues for the condition of the whole ecosystem. The bioindicator has particular
requirements with regard to a known set of physical or chemical variables such that
changes in presence or absence, numbers, morphology, physiology or behavior of that
species indicate that the given physical or chemical variables are outside their preferred
limits. Specifically, bioindicators are a species or group of species that readily reflects
the abiotic or biotic state of an environment, represents the impact of conditions.
Gormot A. (1997) studied about the effect of heavy metals on the growth of
snails. The heavy metals (Chromium, Cadmium, Lead and Zinc) were mixed with soil.
This mixture serves as the food of the snails. Results showed that with cadmium
compared to those of other authors working with earthworms and soil arthropods show
that snails give responses to concentrations comparable to those of earthworms and
much more rapidly and with more sensitivity than those of collembolla..
Ozhan et.al.,(2008) studied the effects of selected environmental factors
on the composition and structure of benthic macroinvertebrate communities in Karakaya

Dam Lake. Results showed that Dreissena polymorpha and Tubifex sp. were the most
abundant benthic macroinvertebratein Karakaya Dam Lake.
Paoletti M. G.(1999) stated that bioindicators are useful where environmental
factors in the past are reconstructed, pesticides and their residues or complex toxic
effluents contain several interacting chemicals and where the environmental factor is
easy to measure but difficult to interpret.
Sencar J. et.al., studied about mosses and some species of mushroom as
bioindicators of radiocaesium contamination and risk assessment. Results showed that
mushroom consumption was not a critical pathway for the transfer of radiocaesium from
fallout to humans after the Chernobyl accident. Mosses, lichens, mushrooms are able to
efficiently accumulate different radioactive elements from their environment to a much
higher degree than other vegetation.
Schilling et.al., (2001) studied about the bioindication of atmospheric heavy metal
deposition in the Southeastern US using the moss Thuidium delicatulum. They use
Lead, Copper, Chromium and Nickel concentrations. The said concentrations were
quantified in the tissue of fern moss. Results showed that Thuidium delicatulum is
worthy of further study as a passive accumulator and bioindicator for ground level
deposition rates of heavy metals in the blue ridge and in the Southern Appalichians.
Stanley et.al.,(2011) said that the formula for mycellial growth was Colony
diameter on the last day in centimeter over the number of days the measurement was
taken after inoculation. The daily mycelia growth was determined using a ruler across

the Petri-dish horizontally. environmental change on a habitat, community or ecosystem

or is indicative of the diversity of a subset of taxa or the whole diversity within an area.

Research Design

METHODOLOGY
8

Gathering of Materials
The Panaeolus antillarum mushroom strains were provided by the Central Luzon
State University Center for Tropical Mushroom. The autoclave, inoculation chamber and
other apparatuses at the Mushroom Center were also used with the supervision of
trained and professional personnel.
Media Preparation
One-eight kilograms of potatoes were peeled,chopped and then boiled in a liter
of water for twenty minutes to produce a potato solution. After twenty minutes, the
potatoes were removed from the solution leaving only the potato concoction. Ten grams
of sugar and twenty grams of colorless bar gulaman were added into the solution to
produce the Potato Sucrose Agar. The mixture was then poured and divided into sterile
bottles and left to cool down and solidify. The bottles were sealed with cotton plugs and
aluminium foil before putting in a refrigerator to preserve the agar.
Heavy Metal Solution Preparation
Two and a half grams of Copper sulfate, Zinc sulfate, and Lead sulfate were
diluted in a liter of distilled water. Concentrations of 100 ppm, 10 ppm and 1 ppm were
prepared for each heavy metal to make up the treatments. A control treatment was also
prepared, composed only of the pure Potato Sucrose Gulaman or PSG. Treatments
were triplicated for more accurate results. The treatments were autoclaved in a water
bath to liquefy at 15 psi for 45 minutes. These were then poured into eighty-three
millimetre disposable petri dishes used in the preparation of the mushroom inoculums.
These were then left inside the inoculation chamber to solidify.
9

Treated Culture Preparation

For the preparation of treated cultures, an eight millimetre in diameter cork borer
was sterilized and used to obtain portions of the P. Antillarum pure culture. The equal
portions of the mushroom strain were then each placed into the center of the petri
dishes with the treated PSGs. The plates were closed and sealed by a one and a half
inch food wrapper on all sides to prevent contamination. The petri dishes were observed
for the growth of the mushroom mycelia.
Measurement of the Mycelial Growth
The mycelial growth of the mushroom inoculums were measured and monitored
in a period of sixteen days starting from the day when development was observed. A
ruler was used to measure the diameter of the mycelia in millimeters (mm) every
twenty-four hours. The data were gathered and recorded.
Calculating for the Percentage Inhibition
The inhibition percentage of the fungus mycelia growth due to metal-containing
compounds and the inhibitory effect of exhibited compound was calculated in relation to
the control using Abottes formula:

I = [(C T)/C] x 100 %

10

Where, I is the percentage inhibition of mycelial growth (%); C is the diameter of

growth measurement of the fungus in control (mm) and T is the diameter growth of the
fungus in treated plates (mm) (Edington et al, 1971).
Determining of Mycelial Growth Rate and Density
The mycelial density was rated as described by Kadiri (1998) as follows:
+
2+
3+
4+
5+

=
=
=
=
=

Very Scanty
Scanty
Moderate
Abundant
Very Abundant

Average linear growth rate (ALG) was calculated by following formula (Elad et al.,
1981):
ALG(mm/day) = (C3-C1)/T
where C3 = Colony diameter in mm after three days, C1= Colony diameter in mm after
one day, T= difference in time (day).

Statistical Analysis
Analysis of Variance (ANOVA) was used to determine if there were significant
differences among the values in the different parameters, namely: density, percentage
inhibition, average linear growth and mycelia growth diameter.

Results and Discussion

11

Results showed that in Treatment 6 (1 ppm Zinc Sulfate) the Panaeolus

antillarum mycelial growth was faster than the other heavy metals concentration in this
study. Treatment 6 (1ppm Zinc Sulfate) 10% average linear growth. The control has the
lowest mycelial growth with a 3.5% average linear growth. Other heavy metals
concentration had the following average linear growth: 100 ppm Lead Sulfate (6.5%), 10
ppm Lead Sulfate (5.335%), 1ppm Lead Sulfate (5.5%), 100 ppm Zinc Sulfate (8.33%),
10 ppm Zinc Sulfate (5.165%), 100 ppm Copper Sulfate (7.165%), 10 ppm Copper
Sulfate (7.25%) and 1ppm Copper Sulfate (7.5%)
Treatment 4 (100 ppm Zinc Sulfate) has the lowest inhibition with a -20.49
mm/day inhibition. The control has the highest inhibition of 0 mm/day. Other heavy
metals concentration had the following inhibition: 100 ppm Lead Sulfate (-3.91
mm/day),10 ppm Lead Sulfate (-7.82 mm/day), 1ppm Lead Sulfate (-9.28 mm/day),
10ppm Zinc Sulfate (-5.85 mm/day), 1 ppm Zinc Sulfate (-16.6 mm/day), 100 ppm
Copper Sulfate (-13.67 mm/day), 10 ppm Copper Sulfate (-18.54 mm/day) and 1ppm
Copper Sulfate (-12.69 mm/day).
Treatment 6 (1 ppm Zinc Sulfate) has the highest density with a 3.67+ density.
The Treatment 7(100 ppm Copper Sulfate) has the lowest density of 1.33 +. Other
heavy metals concentration had the following density: 100 ppm Lead Sulfate (2.67+),10
ppm Lead Sulfate (3+), 1ppm Lead Sulfate (2+), 100ppm Zinc Sulfate (1.67+), 10 ppm
Zinc Sulfate (2.33+), 10 ppm Copper Sulfate(3+), 1 ppm Copper Sulfate (2.5+) and
control(2.67+).
The results of the Analysis of variance showed significant differences among the
values in each group of parameters namely Density, diameter growth, average linear
12

growth and inhibition. This means that the growth of the mushroom reacts differently in
media induced with different heavy metals. Also, these show the varying degrees of
growth and development of the mushroom mycelia in the media contaminated with
heavy metals and the control variable.

13

14

Table 2 Average Linear Growth

Results
(mm/day)
6.5
5.33
5.5
8.33
5.167
10
7.167
8
7.33
3.5

Treatments
T1(100 ppm Lead Sulfate)
T2(10 ppm Lead Sulfate)
T3(1 ppm Lead Sulfate)
T4(100 ppm Zinc Sulfate)
T5(10 ppm Zinc Sulfate)
T6(1 ppm Zinc Sulfate)
T7(100 ppm Copper Sulfate)
T8(10 ppm Copper Sulfate)
T9(1 ppm Copper Sulfate)
Control

Table 2 shows that Treatment 6 (1 ppm Zinc Sulfate) has the highest average
linear growth. And the control has the lowest average linear growth.
Table 3 Inhibition

Treatments
T1(100 ppm Lead Sulfate)
T2(10 ppm Lead Sulfate)
T3(1 ppm Lead Sulfate)
T4(100 ppm Zinc Sulfate)
T5(10 ppm Zinc Sulfate)
T6(1 ppm Zinc Sulfate)
T7(100 ppm Copper Sulfate)
T8(10 ppm Copper Sulfate)
T9(1 ppm Copper Sulfate)
Control

Results
(%)
-3.66
-6.51
-8.39
-18.61
-2.05
-21.46
-11.11
-15.48
-16.78
0

Table 3 shows that Treatment 4 (100 ppm Zinc Sulfate) has the lowest inhibition.
And the control has the highest inhibition.

Table 4 Density
15

Treatments
T1(100 ppm Lead Sulfate)
T2(10 ppm Lead Sulfate)
T3(1 ppm Lead Sulfate)
T4(100 ppm Zinc Sulfate)
T5(10 ppm Zinc Sulfate)
T6(1 ppm Zinc Sulfate)
T7(100 ppm Copper Sulfate)
T8(10 ppm Copper Sulfate)
T9(1 ppm Copper Sulfate)
Control

Density
2.67+
3+
2+
1.67+
2.33+
3.67+
1.33+
2.67+
2.33+
2.67+

Where:
+ to 2+
2+ to 3+
3+ to 4+

=
=
=

Very Scanty to Scanty

Scanty to Moderate
Moderate to Abundant

Table 4 shows that Treatment 6 (1 ppm Zinc Sulfate) has the highest density. And
Treatment 7 (100ppm Copper Sulfate) has the lowest density.

16

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Summary
This scientific investigation was done to determine the mycelial growth
Panaeolus antillarum as bioindicator agent in heavy metal contaminated media. It has
undergone

diameter, average

linear

growth

diameter, inhibition

and

density

measurement. Results showed that in Treatment 6 (1 ppm Zinc Sulfate) the Panaeolus
antillarum mycelial growth was faster than the other heavy metals concentration in this
study. The Treatment 4 (100 ppm Zinc Sulfate) has the lowest inhibition with a -20.49
mm/day inhibition. The control has the highest inhibition of 0 mm/day. The Treatment 6
(1 ppm Zinc Sulfate) has the highest density with a 3.67+ density. The Treatment 7(100
ppm Copper Sulfate) has the lowest density of 1.33 +. Based on the results of the
Analysis of Variance, there were significant differences on the density, percent inhibition,
average linear growth and mycelia growth diameter of the mycelia development of
Panaeolus antillarum, The Panaeolus antillarum mycelia growth varies on its medium so
it can serve as a bioindicator for heavy metal-contaminated media.

17

Conclusions
Based from the results of the experimentation, the following conclusions were
drawn by the researchers:
1. Based on the performed statistical analysis (ANOVA), there were significant
differences in the growth of Panaeolus Antillarum on different heavy metal
concentrations.
2. With 6.5 % in T1; 5.33 % in T2; 5.5 % in T3; 8.33 % in T4; 5.167 % in T5; 10 % in
T6; 7.167 % in T7; 8 % in T8; 7.33 % in T9 and 3.5 % in the Control and with
respect to the result of the statistical analysis, there were significant differences
on the average linear mycelial growth of Panaeolus Antillarum on different heavy
metals concentrations. The highest average mycelial growth was observed in
Treatment 6, with a concentration of 1 ppm Zinc Sulfate and lowest in the
Control. This means that P. antillarum grows better in a Zinc-rich environment.
3. With -3.66 mm/day on T1; -6.51 mm/day on T2; -8.39 mm/day on T3; -18.61
mm/day on T4; -2.05 mm/day on T5; -21.46 mm/day on T6; -11.11 mm/day on
T7; -15.48 mm/day on T8; -16.78 mm/day on T9; 0 mm/day on control, there
were significant differences on the inhibition of Panaeolus Antillarum on different
heavy metals concentrations.
4. With the density of 2.67+ on T1; 3+ on T2; 2+ on T3; 1.67+ on T4; 2.33+ on T5;
3.67+ on T6; 1.33+ on T7; 2.67+ on T8; 2.33+ on T9 and 2.67+ on control, there
were significant differences on the density of Panaeolus Antillarum on different
heavy metal concentrations.

Recommendations
18

The project proponents recommend the following:

1. Use other heavy metals to have more knowledge about Panaeolus Antillarum as
bioindicator.
2. Use other concentrations of heavy metals including higher ppm (parts per
million).
3. Perform and make a follow-up study and experiments to verify results.

Appendices
Appendix A.
I. Materials
19

Inoculation Needle

Face Mask

Alcohol

20

Inoculation Chamber

Cork borer

Gloves

Sugar

Gulaman

21

Potato

Crushing the gulaman bar

Pouring 1 liter of water

Chopping of potatoes

22

Weighing of sugar

Removal of potato from the concoction

Boiling of Potato

23

Boiling of PSG

Concentration Preparation

24

Lead Sulfate

Copper Sulfate

25

Zinc Sulfate

Pouring 1 liter of water

26

Lead Sulfate Solution

Copper Sulfate Solution

Zinc Sulfate Solution

27

Stirring of Solutions

Transferring of solutions into flat bottles

28

29

Concentration

Treated Cultures after 16 days

30

31

32

Petri dish

Culture of Panaeolus (1st petri dish)

Culture of panaeolus (2nd petri dish)

Pouring of PSG (Potato Sucrose Gulaman Agar)

on petri plates
33

Sterilization

Sterilization of cork borer

Inoculation

34

Sealing of Petri dish

Used inoculum (1st petri dish)

Used Inoculum (2nd petri dish)

Labeling

35

Appendix B

Figure 2. Average Linear Growth of Panaeolus antillarum

Average linear Growth(mm/day)

10
9
8
7
6
5
4
3
2
1
0

Average linear
Growth(mm/day)

Figure 3. Inhibition of of Panaeolus antillarum mycelia

Inhibition(%)
0

T1

T2

T3

T4

T5

T6

T7

T8

T9 Control

-5

-10

Inhibition(%)

-15

-20

-25

36

37

38

39

40

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42

43