TOXICITY BIOASSAY WITH BEAN SEEDS (PHASEOLUS VULGARIS)

Javier Alexander SANTISTEBAN VEGA

Universidad Nacional de Ingeniería, Facultad de Ingeniería Ambiental, Escuela Profesional de Ingeniería Ambiental. Av.
Túpac Amaru 210 - Rímac, Lima, Perú, jsantistebanv@uni.pe

TOXICITY BIOASSAY WITH BEAN SEEDS (PHASEOLUS VULGARIS)

ABSTRACT. Wastewater contamination due to dishwashers and detergents is a problem that has been increasing as a result of overcrowding,
among other factors. In this work, the effect of this pollutant on beans (Phaseolus vulgaris) is evaluated in different concentrations (100%,
50%, 25%, 12.5%) from a solution of 100 ml of a dishwasher stock solution and 100 ml of water. With each concentration of dishwasher, the
treatments were carried out by moistening the bean seeds wrapped in cotton in plastic cups; for after 5 days, observe and evaluate its
germination and growth.

BIOENSAYO DE TOXICIDAD CON SEMILLAS DE FRIJOL (PHASEOLUS VULGARIS)

RESUMEN. La contaminación de aguas residuales debido a lavavajillas y detergentes es una problemática que ha ido en aumento a
consecuencia de la sobrepoblación, entre otros factores. En este trabajo se evalúa el efecto de este contaminante sobre frijoles (Phaseolus
vulgaris) en diferentes concentraciones (100%, 50%, 25%, 12.5%) provenientes de una disolución de 100 ml de una solución madre de
lavavajilla y 100 ml de agua. Con cada concentración de lavavajilla se realizaron los tratamientos humedeciendo las semillas de frijoles
envueltas en algodón en vasos de plástico; para después de 5 días, observar y evaluar su germinación y crecimiento.

* Correspondence to: Javier SANTISTEBAN VEGA. jsantistebanv@uni.pe.

INTRODUCTION
During the next 5 decades, the problems related
to the scarcity of water or the contamination of
the vital liquid will affect, without differentiating,
all the inhabitants of the planet, so the world will
need to start looking for solutions in this regard.
However, human beings are not the only ones to
suffer these consequences, since many other
living organisms will also suffer them. Today,
cities dump partially treated and untreated
wastewater into nearby surface and
groundwater. With discharges from industrial
processes, plus the infiltration of fertilizer and
pesticide residues used in agriculture, household
waste and others, the pollutant load increases.
The result is that only about a third of the
potential resource, probably about 12,500 km3
per year, can be used for people's needs, a
proportion that is decreasing as pollution
increases (WHO 2013).
It is estimated that more than five million people
die annually from diseases linked to the
consumption of contaminated water,
inadequate sanitation and rudimentary hygiene.
Human health depends on a safe and therefore
safe water supply and reliable sanitation
services, but as mentioned, organisms such as
plants and animals are also affected. For this
reason, immediate measures are required.
A strategy that supports the investigation of
these pollutants are ecotoxicological bioassays,
measuring their effect on the growth of
bioindicator organisms; Among them, terrestrial
plants present representative species of
horticultural agroecosystems. Sprouted seed
phytotoxicity tests are simple, versatile and
useful for evaluating the toxicity of waters,
sediments and soil samples. Some plant species
have advantages over other biological organisms
for the development of bioassays, such as: being
able to be stored in seed form for a year or more,
minimal maintenance cost, samples do not
require aeration, samples with high turbidity do
not require Additional filtration and finally tests
can be carried out without pH adjustment.
The three most important characteristics of tests
with terrestrial plants is that they can be used
with colored or cloudy samples, in static, semi-
static and continuous flow tests, and with a
minimum cost of maintenance in the laboratory
(Wang, 1991). The use of the seeds of terrestrial
plants as ecotoxicological tools is advantageous,
since it requires little sample volume, compared
to other organisms that require a greater

405
Javier Alexander SANTISTEBAN VEGA

quantity. The analysis of the results of this complement REAL STATISTICS, ANOVA tests and
bioassay was performed using the statistical multiple comparisons.

EXPERIMENTAL • Treatment 3 (25%): 100 ml of Treatment

2 + 100 ml of water
1. Materials and Methods • Treatment 4 (12.5%): 100 ml of
• Organism: Phaseolus vulgaris Treatment 3 + 100 ml of water
• Contaminant: dishwashing solution B. Each paper towel will be moistened with
• Negative control: Tap water a different solution to wrap 5 bean
• Disposable cups seeds, these will be constantly
• Paper towel moistened in each cup.
• Cotton C. 5 replicates will be made for each
• Dropper solution and 3 for the negative control
• Tanks (jug and glasses with measure) (200 ml of tap water).
2. Process D. After 120 hours, the growth will be
A. 4 solutions with different concentrations observed and the% germination will be
will be prepared: calculated, as well as the measurements
• Treatment 1 (100%): 100 ml of stock of the roots.
solution + 100 ml of water E. Comparison with the negative control.
• Treatment 2 (50%): 100 ml of Treatment RESULTS AND DISCUSSIONS
1 + 100 ml of water
Table 1: Measurements after 120 days (cm)

NC 12.50% 25% 50% 100%

11.3 7.3 5.2 6.5 1.5
10.8 0.9 8.1 4.5 3.7
11 2.3 7.6 7.9 4.5
11.2 2 7.5 5.8 2.5
11 0.9 7 1.6 3.6
10.5 12.2 6.6 4.8 3.9
10.2 7.6 6.8 5.3 3
10.3 8.1 7.3 6.6 5.7
10.6 8.5 7.9 4.8 5.1
9.7 8.5 6.6 5.2 5
11.1 13.3 6.2 4.8 2.9
10.5 10.5 6.5 6.3 4.7
10.3 6.2 7.2 7.7 4.8
10.6 6.8 6.7 7.7 3
10.1 0.9 6.3 7.3 3.5
6.2 6.3 5.5
6.6 7.7 3.1
7.1 6.5 3.4
6.7 6.9 4.2
6.7 7.3 4.9

406
Leather and Footwear Journal 20 (2020) 4
 TOXICITY BIOASSAY WITH BEAN SEEDS (PHASEOLUS VULGARIS)

3. Annova test

Table 2: Statistical data of the Annova test

DESCRIPTION Alpha 0.05
Group Count Sum Mean Variance SS Std Err Lower Upper
Group 1 15 159.2 10.6133333 0.20552381 2.87733333 0.56595739 9.48878805 11.7378786
Group 2 21 12942.50% 616.31% 13.9043512 278.087024 0.4783213 5.212681 7.11350947
Group 3 21 13845% 659% 2.56207143 51.2414286 0.4783213 5.64244291 7.54327138
Group 4 16 8730% 546% 4.29329167 64.399375 0.54798589 4.36741371 6.54508629
Group 5 21 7950% 379% 1.55028571 31.0057143 0.4783213 2.83530005 4.73612852

Table 3: Statistical data of the Annova test (Part 2)

Sources SS df MS F P value Eta-sq RMSSE Omega Sq
Between
Groups 425.382403 4 106.345601 22.1340453 1.0469E-12 0.49869373 1.15179719 0.47349608
Within
Groups 427.610875 89 4.80461657
Total 852.993278 93 9.17197073

• It is observed that P value <0.05.

• At least one treatment is different.
4. Box-Plot

Illustration 1: Box-Plot of the treatments

5. Normality Test

Table 4: Statistical data of the Normality test

Shapiro-Wilk Test
CN 0.125 0.25 0.5 1
W-stat 0.96832899 0.91630271 0.96838123 0.91035794 0.97109513
p-value 0.83258202 0.08412726 0.72038869 0.13713197 0.77782048
alpha 0.05 0.05 0.05 0.05 0.05
normal yes yes yes yes yes

407
Revista de Pielărie Încălţăminte 20 (2020) 4
Javier Alexander SANTISTEBAN VEGA

• The existence of a normality was 6. Dunnett test

determined as each treatment resulted
in "yes".
Table 5: Statistical data of the Dunett test

DUNNETT'S TEST alpha 0.05

group mean size ss df d-crit
Group 1 10.6133333 15 2.87733333
Group 2 616.31% 21 278.087024
Group 3 659% 21 51.2414286
Group 4 546% 16 64.399375
Group 5 379% 21 31.0057143
94 427.610875 89 2.48614607

Table 6: Statistical data from the Dunett test (Part 2)

D-TEST
group mean std err d-stat lower upper p-value mean-crit Cohen d
Group 2 445.02% 0.74101217 6.00562079 2.6079736 6.29250259 0 1.84226449 2.03027038
Group 3 402.05% 0.74101217 5.42565473 2.1782117 5.86274068 0 1.84226449 1.83420607

Group 4 515.71% 0.78777935 6.54635504 3.1985488 7.11561787 0 1.95853454 2.35274458

Group 5 682.76% 0.74101217 9.21390946 4.98535455 8.66988354 0 1.84226449 3.11486991

• In case of being d-stat> d-scrit it is At 95% confidence, there are differences

concluded that there is a difference between the values of the negative
between the treatments and the control, control and the third test.
it is for this reason that:
At 95% confidence there are differences
At 95% confidence, there are differences between the values of the negative
between the values of the negative control and the fourth test.
control and the first test.
7. Tukey's test
At 95% confidence there are differences
between the values of the negative
control and the second test.

Table 7: Statistical data of the Tukey test

TUKEY HSD/KRAMER alpha 0.05

group mean n ss df q-crit
Group 1 10.6133333 15 2.87733333
Group 2 616.31% 21 278.087024
Group 3 659% 21 51.2414286
Group 4 546% 16 64.399375
Group 5 379% 21 31.0057143
94 427.610875 89 3.93789888

408
Leather and Footwear Journal 20 (2020) 4
 TOXICITY BIOASSAY WITH BEAN SEEDS (PHASEOLUS VULGARIS)

Table 8: Statistical data of the Tukey test (Part 2)

Q TEST
group 1 group 2 mean std err q-stat lower upper p-value mean-crit Cohen d
Group 1 Group 2 4.4502381 0.52397473 8.49323037 2.38687859 6.5135976 4.0326E-07 2.0633595 2.03027038
Group 1 Group 3 4.02047619 0.52397473 7.67303451 1.95711669 6.08383569 4.8388E-06 2.0633595 1.83420607

Group 1 Group 4 5.15708333 0.55704412 9.25794409 2.96349991 7.35066675 3.6452E-08 2.19358342 2.35274458

Group 1 Group 5 6.82761905 0.52397473 13.0304357 4.76425954 8.89097855 3.0143E-13 2.0633595 3.11486991
Group 2 Group 3 0.4297619 0.4783213 0.89847955 -1.453819 2.31334281 0.96889372 1.88358091 0.19606431
Group 2 Group 4 0.70684524 0.51433442 1.37429113 -1.3185517 2.73224219 0.86715818 2.02539695 0.3224742
Group 2 Group 5 2.37738095 0.4783213 4.97025944 0.49380005 4.26096186 0.00610811 1.88358091 1.08459953
Group 3 Group 4 1.13660714 0.51433442 2.20986014 -0.8887898 3.1620041 0.52512062 2.02539695 0.51853851
Group 3 Group 5 2.80714286 0.4783213 5.86873899 0.92356195 4.69072376 0.0007106 1.88358091 1.28066384
Group 4 Group 5 1.67053571 0.51433442 3.24795626 -0.3548612 3.69593267 0.15563395 2.02539695 0.76212533

• In case of being q-stat> q-scrit then there • “Group 5” could be eliminated, since
is a difference between the examined when compared with “Group 4” its
treatments (at 95% confidence there is a values do not present such distant
significant difference). Therefore, it was values. However, the decision was made
observed that in the first 4 to keep it, since it is the treatment with
concentrations they generate the highest concentration.
differentiated effects. 8. Percentage of Inhibition and Cl 50

Table 9: Average of each Treatment

CN 0.125 0.25 0.5 1

Mean 10.6133333 6.465 6.91 5.78666667 3.925

Table 10: Percentages of inhibition of each treatment

Porcentaje
de
Inhibición
12.50% 39.0860553
25% 34.8932161
50% 45.4773869
100% 63.0182161

409
Revista de Pielărie Încălţăminte 20 (2020) 4
Javier Alexander SANTISTEBAN VEGA

Illustration 2: Percentage of inhibition vs Concentration of each treatment

• Replacing in the equation of the line the
value of Cl 50 is obtained, which was 61.1
• R favorable for the bioassay.
CONCLUSIONS
• Treatment 1 shows the least growth
compared to the other treatments.
• A brown color was observed in the roots
and stems of the beans, except for the
negative control.
• The Cl 50 resulted in a concentration of
61.1%.
• The percentage of inhibition in
treatment 1 was higher than that of
Treatment 1, contrary to what was
originally thought.
toxicidad sobre toxicidad sobre semillas
de lechuga (Lactuca Sativa L.) 2010.
2. Rodríguez Romero, Alexis; Robles
Salazar, Cristopher; Ruíz Picos Ricardo;
López López, Eugenia; Sedeño Díaz,
Jacinto; Rodríguez Dorantes, Angélica.
Escuela Nacional de Ciencias Biológicas,
Laboratorio de Bioconservación y
Manejo, Instituto Politécnico Nacional,
Prol. de Carpio y Plan de Ayala s/n, Col.
Sto. Tomás, Mexico, D.F., 11340 Mexico.
Índices de germinación y elongación
radical de Lactuca sativa en el
biomonitoreo de la calidad del agua del
río Chalma. 2013.

REFERENCES
1. Pérez Oyola, Francy; Baracaldo Cubides,
César. Universidad de La Salle, Bogotá.
Determinación de la concentración de
inhibición media (CE50-120) producida
por la plata (Ag+) y los detergentes
aniónicos (Las) mediante bioensayos de

410
Leather and Footwear Journal 20 (2020) 4