Remember- we need to centrally focus on a biological question. This seems political to me? If you want to do research on abortion then maybe look at the psychological aspects (development

Impact on Freshwater Organisms from Synthetic Textile Dyes

Angie Lee

Introduction

Textile industries involve converting raw materials such as wool, cotton, or linen into

finished products such as cloth and yarn. They produce large scales of numerous forms of fabrics

and threads for colored clothing and accessories for individuals at home. These industries also

use textile products to create synthetic livers and arteries for transplant patients (Sivaram et al.,

2019). Textile industries are found worldwide and are the leading resource of employment and

economic market (Lellis et al., 2019). These industries are significant to consumers but are the

primary cause of the high consumption of chemicals and fuels and are the largest global polluters

(Lellis et al., 2019). India, China, the European Union, and the United States of America are the

leading countries for the dyestuff sector (Moorthy et al., 2020). Dyestuff refers to organic

substances which yield colored materials or products.

India, for example, is one of the top leading countries in the textile industry because its

central role in the Indian economy. Their textile industries rely on cotton making, which is sixty-

five percent of the raw materials in the country (Kumar et al., 2019). As a result, India

contributed about one hundred thousand tons of dyestuff (Moorthy et al., 2020). The textile

sector gains about twenty-seven percent of foreign exchange earnings and provides twenty-one

percent of employment. Thus, the Indian textile industry depends on cotton production, which

consists of the apparel-making enterprise.

The global textile industries are responsible for the widespread environmental impacts,

including the effects on water bodies (Lellis et al., 2019). These industries are leaking untreated

sewage into these waterways, which leads to most residual waters having high concentrations of

biochemical oxygen demand, chemical oxygen demand, and large amounts of non-biodegradable

organic compounds from textile dyes (Lellis et al., 2019). In recent years, the amount of

chemical compounds found in aquatic environments has increased. One of the chemical

compounds, synthetic organic dyes, is identified as a containment in water bodies. Synthetic

organic dyes are micropollutants because of their low concentration, from one nanogram per liter

to one microgram per liter. Due to the large production of textile dyes, synthetic organic dyes are

in many application areas. This determines that the dyes are ubiquitous in water bodies (Tkaczyk

et al., 2020).

High amounts of textile dyes found in wastewater are non-biodegradable or slow

biodegrading, making clean-up difficult for sewage treatment plants (Tkaczyk et al., 2020). In

wastewater, the initial cleaning of raw fibers leaves high volumes of impurities. The usage of

detergents removes these impurities but can lead to the risk of being discarded into freshwater

environments (Stone et al., 2020). The leaked dyes cause physical damage to water bodies and

prevent light penetration through water for photosynthesis of aquatic plants, which causes

oxygen deficiency (Lellis et al., 2019). Moreover, synthetic dyes have mutagenic, genotoxic, and

carcinogenic effects (Gita et al., 2019). These agents spread among the aquatic environment and

cross the entire food chain. There are cases of biomagnification in which higher trophic levels

show larger contamination amounts than their preys (Lellis et al., 2019).

Textile dyes and other industrial pollutants are ultimately toxic and carcinogenic in

aquatic environments, particularly freshwater. Products from dyes by biodegradation can be

harmful because of the ability to produce aromatic compounds (Moorthy et al., 2020). The dyes

are water soluble and can absorb into the skin of aquatic organisms (Zohoorian et al., 2020). One

study measuring the impact of indigo dye on microorganisms such as Scenedesmus quadricauda

noted both growth reduction and biomass production. The buildup of biomass suggests the

removal of nutrients such as phosphate and nitrate from wastewater and can exploit biofuels

(Oyebamiji et al., 2019). Moreover, the microscopic results indicated morphological changes of

the algae from exposure to the dye. The study also follows the exposure of the dye to the health

of aquatic animals such as Catla catla. The Catla catla resulted in micronuclei development and

histopathological changes (Moorthy et al., 2020). This study suggests a significant concern

around the release of textile dyes in freshwater environments.

There are not many studies relating to textile dyes in aquatic environments. Many studies

focus on the toxicity of heavy metals and pesticides in aquatic environments. However, synthetic

dyes’ discharge in these environments is more toxic to several aquatic microorganisms (Gita et

al., 2018). A few researchers conducted studies on textile dyes on microalgae in freshwater

environments (Gita et al., 2021). Algae is more sensitive to pollutants compared to other aquatic

organisms. The presence of synthetic dyes in algae affects the biochemical parameters such as

pigments content, growth, and other nutrients (Yusuf 2019). Since textile dyes do not have

effective data on aquatic species, we can determine which synthetic dyes affect certain species.

The micropollutant burden impacts various aquatic organisms in their environments.

Synthetic dyes found in the hydrosphere result in environmental degradation because some

aquatic species do not exhibit growth, and there are lower chances of photosynthesis (Gita et al.,

2021). In other words, the influence of the toxicity of textile dyes causes ecological changes in

aquatic environments, which result in a negative impact on the health of aquatic organisms. This

study aims to further investigate the impact of toxicity of commonly used synthetic dyes on

freshwater aquatic organisms, specifically aquatic algae, since their species are more sensitive to

textile dyes. It is also a great source to begin with the primary producers because of the

biomagnification correlation with the entire food chain. The ecological concern among an

organism increases as toxins increase in wastewater, which can significantly impact the health of

organisms. This will help provide adequate information to reduce the impact on freshwater

environments.

While there are few studies on the impact of textile dyes on aquatic environments, these

textile dyes could potentially be more significant in the upcoming years because of the increased

production of colored clothing and accessories due to our population growth (Gita et al., 2017).

This study can help textile industries change their policy of untreated wastewater discharge to

water bodies. This study can also help further investigate the impacts on animals and humans

exposed to these micropollutants or living near aquatic environments. Toxicity in dyes can

impact any living organism. Synthetic dyes are not commonly known as a pollutant because of

their small size, but they could potentially be a pollutant factor.

Methods

Algal Selection

We will collect algal species samples at four selected sampling sites. We will decide to

collect from four sample sites to get a chance to get more than one species. The four sampling

sites will be freshwater areas near urban and textile industrial zones (Methneni et al., 2020).

Once the four water samples with the selected algal species are in their designated tubes, they all

will be transported to the laboratory and contained in a container of ice to keep cool. We will

determine our algal species collected using one drop of each sample on a glass slide to use with a

light microscope in the lab. The two most abundant microalgae near those sampling sites will be

collected and examined to be Chlorella vulgaris and Spirulina platensis.

Isolation of Microalgae

We will isolate Chlorella vulgaris and Spirulina platensis from the freshwater samples

using standard plating methods to break up algal populations (Lee et al., 2014). We will

subculture the pure culture of Chlorella vulgaris in sterile BG-11 media and Spirulina platensis

in sterile modified Nallayam Research Centre medium (Moorthy et al., 2020). We will use thirty

Erlenmeyer flasks to place each culture and its equivalent media (Chlorella vulgaris and

Spirulina platensis will have fourteen experimental flasks and one control flask each). Both

cultures will be under photoautotrophic conditions and grown in a growth chamber using

fluorescent lamps. We will monitor each group by shaking each flask every morning and night,

keeping them at a temperature around 24 degrees Celsius, and placing them in sixteen hours of

light and eight hours of dark periods (Gita et al., 2019).

Experimental Design

We will use the seven most common textile dyes found in cotton, linen, and wool. We

will use acid and reactive dyes: Optilan red, Optilan yellow, Lanasyn brown, Lanasyn olive,

Drimarene red, Drimarene blue, and Methylene blue. We will add about 0.1 to 100 milligrams

per liter of each of the seven dyes into the following fourteen experimental flasks containing 100

milliliters of either Chlorella vulgaris or Spirulina platensis (Moorthy et al., 2020). To determine

the toxicity of assay, we will follow the OECD guidelines, so the concentration of each dye will

change when necessary. The fourteen experimental flasks for Chlorella vulgaris and Spirulina

platensis will have two different dosage sets (the first group of seven flasks and the second group

of seven flasks for each species will have different dye amounts added) of Optilan red, Optilan

yellow, Lanasyn brown, Lanasyn olive, Drimarene red, and Drimarene and Methylene blue. The

first set of the seven flasks for Chlorella vulgaris will use 0.1, 1, 10, 30, 50, 80, 90 milligrams

per liter dosages, respectively. The following second set of dye dosages will use 30, 40, 50, 60,

70, 100, 80 milligrams per liter. The fourteen experimental flasks for Spirulina platensis also

will have the same corresponding dosage sets to each dye like Chlorella vulgaris. Incubation will

occur for all experimental flasks (containing the specific culture with the measured dye solution)

and the two control flasks in the growth chamber for a week. We will use algal culture density at

3 x 105 cells per milliliter throughout the experiment (Gita et al., 2019).

Data Analysis

We will observe the experimental and control flasks of the two species for growth

inhibition each night. We will determine the growth of both cultures by taking a small amount of

each sample and counting the number of cells using the Sedgewick Rafter and a light

microscope. On the other hand, all fourteen experimental flasks for both species will have five

milliliters of culture measured out to be centrifuged at 5000 ppm for about ten minutes at four

degrees Celsius (Moorthy et al., 2020). Using a refrigerated centrifuge, we will collect fresh algal

culture to estimate the growth inhibition rates of pigment content. We will use a Double beam

UV-visible spectrophotometer to measure the turbidity of each fresh algal culture collected (Gita

et al., 2020). We will discard the supernatants collected in each tube in the experiment. We will

wash the remaining pellets from the seven sample sets of Chlorella vulgaris and Spirulina

platensis with sterile distilled water to rinse off any absorbed dye on the algae surface. We then

will add a selected 0.05 M buffer at a pH of 6 to the pellets and place them under ice. After

taking each sample out of the ice, we will collect the homogenate by using the centrifuge at the

same settings. We will measure the optimal densities at wavelengths of 460, 664, and 652

nanometers and observe the results by comparing the concentration of pigment of chlorophyll a

and b and carotenoid in Chlorella vulgaris and Spirulina platensis.

The results we expect are to identify if higher concentrations of each dye will cause

higher growth inhibition than lower concentrations. We will also compare the control groups to

compare the inhibition of growth of both cultures. All the data collected will be inputted into

Excel to compare the seven days of the concentration-dependent method of Chlorella vulgaris

and Spirulina platensis from each experimental flask with their corresponding dye amounts. We

can determine the layout of the different concentration dyes, including the control group, and see

the potential decrease in the growth of each microalgae species. Moreover, using excel will

represent the data on the pigment content of the two species. We hope to see that higher dye

concentrations decrease pigment content in Chlorella vulgaris and Spirulina platensis. Thus, the

results collected will help identify that higher concentrations of textile dyes in freshwater

environments can impact the health of microalgae and other organisms near these environments.

References

Gita, S., Hussan, A., Choudhury, T.G. 2017. Impact of Textile Dyes Waste on Aquatic

Environments and its Treatment. Environment and Ecology. 35(3C): 2349-2353.

Gita, S., Shukla, S.P., Prakash, C., Saharan, N., Deshmukhe, G. 2018. Evaluation of Toxicity of

a Textile Dye (Optilan Red) towards a Green Microalga Chlorella vulgaris. International

Journal of Current Microbiology and Applied Sciences. 7(8): 2246-3355.

Gita, S., Shukla, S.P., Deshmukhe, G., Choudhury, T.G., Saharan, N., Singh, A.K. 2021.

Toxicity Evaluation of Six Textile Dyes on Growth, Metabolism and Elemental

Composition (C, H, N, S) of Microalgae Spirulina platensis: The Environmental

Consequences. Bulletin of Environmental Contamination and Toxicology. 106, 2: 302-309.

Gita, S., Shukla, S.P., Saharan, N., Prakash, C., Deshmukhe, G. 2019. Toxic Effects of Selected

Textile Dyes on Elemental Composition, Photosynthetic Pigments, Protein Content and

Growth of a Freshwater Chlorophycean Alga Chlorella vulgaris. Bulletin of Environmental

Contamination and Toxicology. 102, 6: 795-801.

Kumar, P.S., Pavithra, K.G. 2019. Water and Textiles. Woodhead Publishing. 21-40.

Lee, K., Eisterhold, M.L., Rindi, F., Palanisami, S., Nam, P.K. 2014. Isolation and screening of

microalgae from natural habitats in the midwestern United States of America for biomass

and biodiesel sources. Journal of Natural Science, Biology and Medicine. 5, 2: 333-339.

Lellis, B., Favaro-Polonio, C.Z., Pamphile, J.A, Polonio, J.C. 2019. Effects of textile dyes on

health and the environment and bioremediation potential of living organisms.

Biotechnology Research and Innovation. 3,2: 275-290.

Methneni, N., González, J.A.M., Van Loco, J., Anthonissen, R., de Maele, J.V., Verschaeve, L.,

Fernandez-Serrano, M., Mansour. H.B. 2020. Ecotoxicity profile of heavily contaminated

surface water of two rivers in Tunisia. Environmental Toxicology and Pharmacology. 82:

103550.

Moorthy, A.K, Govindarajan, R.B., Shukla, S.P., Kumar, K., Bharti, V.S. 2021. Acute toxicity of

textile dye Methylene blue on growth and metabolism of selected freshwater microalgae.

Environmental Toxicology and Pharmacology. 82: 103552.

Oyebamiji, O.O., Boeing, W.J., Holguin, F.O., Ilori, O., Amund, O. 2019. Green microalgae

cultured in textile wastewater for biomass generation and biotoxification of heavy metals

and chromogenic substances. Bioresource Technology Reports. 7: 100247.

Sivaram, N.M., Gopal, P.M., Barik, D. 2019. Toxic Waste From Textile Industries. Woodhead

Publishing. 43-54.

Stone, C., Windsor, F.M., Munday, M., Durance, I. 2020. Natural or synthetic – how global

trends in textile usage threaten freshwater environments. Science of The Total Environment.

718: 134689.

Tkaczyk, A., Mitrowska, K., Posyniak, A. 2020. Synthetic organic dyes as contaminants of the

aquatic environment and their implications for ecosystems: A review. Science of The Total

Environment. 717: 137222.

Yusuf, M. 2019. Synthetic Dyes: A Threat to the Environment and Water Ecosystem. Textile and

Clothing: Environmental Concerns and Solutions. 11-26.

Zohoorian, H., Ahmadzadeh H., Molazadeh, M., Shourian, M., Lyon, S. 2020. Microbial

bioremediation of heavy metals and dyes. Handbook of Algal Science, Technology and

Medicine. 659-674.

Research Proposal Components

Background 1. The background should be a big-picture assessment of the topic that you have selected to study

and be somewhere between 3 – 4 pages. The background includes relevant information that

ultimately leads to justifying why you are proposing to conduct your research. Recall that you will

not be doing the research – merely proposing that it be done. The conclusion of the background

section must include the specific hypothesis/question you are addressing. Some researchers list

Specific Aims at the end of their background sections when they are writing proposals. You may

wish to do this. You also will have the rubric to work with ahead of time. This is will help you craft

your background section.

2. Please include all references you have already found and please be sure to use them in the text

with in-text citations. You may add more references than the original five.

Methods 1. Your proposal methods should be a clear description of how you will test your hypothesis or

answer your research question. Page limit = 2 – 3 pages. Your methods likely will be similar to

techniques used by other researchers. You must cite those methods. Your methods also must be

repeatable. Can a reader follow your technique and repeat your study later on? If not, then you

must revise your writing so it is clear. Your methods cannot be in a series of steps – listed out as

1, 2, 3, and so on. This section also must be written in paragraph form. Finally, what results do

you expect to get? What will be the significance of those results? How will you use the

information?

2. Please include all references that are relevant to the methods you want to use. You may need to

add even more sources – that is fine! Please add all that you need- but remember to use all the

references you cite. Continue to use in-text citations.

References 1. All references must be in alphabetical order.

2. Citation format must be consistent throughout the document.

3. All references must be used within the body of the background and methods.