The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
The Effect of Select Endocrine
Disruptors on the growth of
Lemna minor (Duckweed) and
mortality rate of Artemia NYOS
(Sea Monkeys)
Candidate Name: Yeyoung (Romy) Lee
Candidate Number: kbc377
Subject: Biology
Word Count:
1
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Contents:
Content
Page(s)
Title
1
Contents
2
Research Question & Hypothesis
3
Introduction
3
Background Information
3-9
Methodology 1: Solution Making
9-11
Investigation 1: EDC and Duckweed
- Methodology 2
- Results & Data Analysis
11-12
12-20
Investigation 2: EDC and Sea Monkeys
- Methodology 3
- Results & Data Analysis
20-21
21-25
Discussion
25-26
Conclusion
26-27
Appendix
27-29
Bibliography
30-32
2
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Introduction
Research question:
How are the growth of duckweed (lemna minor) and mortality of sea monkeys (artemia NYOS) present in
aquatic environments impacted via contamination with varying concentrations of endocrine disrupting
chemicals Bisphenol A (BPA) and Triclosan (TCS) over 7 to 11 day periods?
Two separate experiments were conducted under similar conditions and methodology, in order to answer
this question.
●
●
The effect of BPA and TCS on the growth and budding of Duckweed
The effect of BPA and TCS on the mortality rate of Sea monkeys
Hypothesis: Increasing concentrations of both endocrine disrupting chemicals BPA and TCS will
demonstrate linear or exponential correlation with the growth of lemna minor (duckweed) and artemia
NYOS (sea monkeys).
Background Information
Endocrine disruptors
According the European Union’s 'Community strategy for endocrine disruptors' (European Commission),
an endocrine disruptor (EDC) is “an exogenous substance that alters function(s) of the endocrine system
and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations”.
EDCs are simultaneously associated with altered reproductive function, breast cancer, abnormal growth
patterns, neurodevelopmental disruptions, as well as abnormalities in the immune system. (Monneret,
2017) Currently, most evidence that suggests such effects of EDC comes from global reports and studies
regarding non-human wildlife species such as crustacea, fish, reptiles, birds and mammals.
(Directorate-General for Environment) However, there is limited evidence to whether EDCs can cause the
same effects in humans. (Directorate-General for Environment) Despite this, the potential impacts of
EDCs may pose a significant threat to public health as even at low doses they may lead to life-long effects
and intergenerational damage.
There are several mechanisms through which EDCs may function in the body. Bisphenol A (BPA) is
regarded as a weak agonist (Gao et al., 2015) to oestrogen receptor β; an agonistic chemical mimics the
hormone and binds to its cellular receptor, initiating unwarranted (ie. at the wrong time or in an excessive
manner) normal responses to the naturally occurring hormone. (Directorate-General for Environment)
Triclosan (TCS) has been found to interfere with the synthesis and breakdown rates (metabolic processes)
of thyroid hormones (Homburg et al., 2022), as well as displacing hormones from hormone receptors
(antagonist) and disrupting enzyme activity in the production of steroid hormones. (Wang et al., 2015)
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The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
One significant category of potential EDCs is man-made chemicals such as pesticides, consumer and
medical products, industrial chemicals, as well as their respective by-products released into the
environment. (Directorate-General for Environment) Both BPA and TCS belong in this category. Below is
a table of some other EDC chemicals and their common sources.
Table 1: EDCs and their common sources (Endocrine Society, 2021)
Common EDCs
Used in
DDT, Chlorpyrifos, Atrazine, 2, 4-D, Glyphosate
Pesticides
Lead, Phthalates, Cadmium
Children’s products
Polychlorinated biphenyls (PCBs) and Dioxins
Industrial Solvents or Lubricants and their
Byproducts
Bisphenol A (BPA), Phthalates, Phenol
Plastics and Food Storage Materials
Brominated Flame Retardants, PCBs
Electronics and Building Materials
Phthalates, Parabens, UV Filters
Personal Care
Sunscreen
Triclosan
Anti-Bacterial Soaps, Colgate Total
Perfluorochemicals
Textiles, Clothing, Non-Stick Food Wrappers,
Microwave Popcorn Bags, Old Teflon Cookware
Products,
Medical
Tubing,
There are several exposure pathways through which EDC may contaminate organisms, such as
the ingestion of food, dust and water, inhalation of gases, and direct contact on skin. (Monneret, 2017)
Once these chemicals enter the food chain through these different pathways, their highly lipophilic nature
allows accumulation in adipose tissue (and thus a relatively long half-life within the bodies of organisms),
meaning humans and other predators at the top of the food chain may be exposed to stronger doses of
EDC due to bioaccumulation and bioamplification. (Lauretta et al., 2019)
One significant pathway of EDC contamination is the ingestion of EDC polluted water or aquatic
species: this is the main pathway that will be investigated in this paper, where the impact of the EDCs
Bisphenol A and Triclosan will be observed in two different aquatic inhabitants- lemna minor (duckweed)
and artemia NYOS (sea monkey). Below is a flow chart depicting this contamination pathway. (Silva et
al., 2018)
4
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Figure 1: Routes of contamination of water bodies and exposure of fish and/or amphibian to substances
characterized as endocrine disruptors (STS = Sewage Treatment Station; EDs = Endocrine disruptors)
(Adapted from Aquino et al., 2013).
Some barriers to existing knowledge on EDCs include complex dose-effect relationships (meaning the
chemicals cause diverging effects at different concentrations, and the trend is not necessarily visible) and
large windows of impact (it is difficult to determine the timing of exposure accurately, as it takes a long
time to observe the effects of EDCs) (Lauretta et al., 2019), which both hinder the formation of any
accurate conclusions regarding these hazardous substances.
Bisphenol A (BPA)
Bisphenol A (BPA) is a synthetic oestrogen (EWG) which mimics the female sex hormone 17β-estradiol
(NZFA), and forms various materials such as hard polycarbonate plastic, thermal receipt paper, epoxy
resins, the protective lining of food and beverage containers, industrial equipment and piping, as well as
sealants in construction and dentistry. (EWG) Due to its widespread use and readily leaching nature, BPA
has become a ubiquitous pollutant and a threat to wildlife in different environments. (EWG) In New
Zealand, however, the average dietary exposure to BPA via consumption of canned foods is estimated as
0.008 µg/body weight (kg)/day, which is well below the legal standard assigned in Europe of 50 µg/body
weight (kg)/day (NZFA). The use of BPA remains completely unregulated in New Zealand. (dashboard,
2019)
Despite this, studies suggest a range of significant health consequences of BPA exposure (EWG),
including cerebral impairments (behavioral, learning and memory), cardiovascular abnormalities,
diabetes, obesity, reproductive cancer, thyroid and sex hormone disruption, changes to egg and sperm
development and fertility, and genetic alterations that can be passed on to future generations. It is also
5
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
known that pregnant women, infants and children are most susceptible to the hazards of BPA exposure, as
hormonal disruption can affect the routine growth and development essential during these periods (EWG).
The main effects of interest for BPA are developmental and reproductive toxicity (NZFA) , as it has been
found to cause adverse reproductive outcomes in non-human animals. Specifically, it has been
demonstrated that BPA disrupts the reproductive axis in mice, rats, and zebrafish. Collectively, such
studies show that BPA exposure affects the reproductive tract and reduces non-human animal fertility.
(Gonsioroski et al., 2020) Although such evidence suggests similar correlations in human biological axes
(ie. BPA association with impaired reproductive function in men and women), this yet to be confirmed.
Two main factors contribute to this ambiguity; primarily, BPA is metabolised very differently in humans
than in other animals such as rodents. In humans, BPA is rapidly converted into and excreted as a
non-estrogenic substance, mostly via urine. In rodents, BPA is also converted into different forms but then
are recirculated and converted back into free BPA, meaning there are higher levels of immediate
accumulation and circulation of BPA in non-human organisms, which in turn would produce more
significant consequences then in humans. (NZFA)
This is supported by the second main factor; the studies conducted on these experimental animals have
shown effects only at exposure levels far in excess of those present in humans. However, some have
shown effects at very low doses, including effects on organ weights, tissue architecture, receptor
expression and behaviour. (NZFA) At higher dose levels of exposure, it has been linked with other more
severe effects such as liver toxicity and reproductive toxicity. (NZFA) Similar research has also been
conducted regarding aquatic environments, as other prominent pollution sites of BPA are rivers, effluent
from sewage treatment plants, and water from water treatment plants. (Gonsioroski et al., 2020) This is
relevant to the topic under investigation in this essay - the effect of BPA on the flora and anima in aquatic
environments.
It is important to note that previous research mainly revolves around BPA exposures that span within 24
hours of initial exposure. However, it is essential that the long term impacts of BPA exposure are also
taken into account-especially due to the low rate of accumulation in organisms as discussed earlier. One
example of such long term consequences of BPA exposure is the potential for trophic transfers; despite
conflicting evidence on the bioaccumulation of BPA in aquatic organisms, the fact of whether BPA
displays trophic transfer is yet to be determined. However, a study by Ishihara and Nakajima in 2003
suggests that BPA can accumulate in zooplankton via phytoplankton. This conclusion is grounded on the
observation of BPA recovery in water and marine phytoplankton as well as medium sized zooplankton.
(Corrales et al., 2015) Among the species studied by Ishihara and Nakajima was Artemia sp., which
recovered >80% of the BPA released in their controlled environment. (Corrales et al., 2015) This aspect
of potential for trophic transfer is in close relation to the relevance of this investigation, as it observes the
impact of BPA exposure on artemia NYOS - a close relative of the Artemia sp - and therefore highlights
the potential biotoxicity that will be generated if trophic transfer were to be successful.
6
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Triclosan (TCS)
Triclosan (TCS) is a preservative and anti-bacterial agent and a potential endocrine disruptor used mainly
for personal hygiene and disinfection purposes (Carey et al. , 2015). It has some medical applications, as
well as functioning as a bacteria-resisting agent in products including garbage bags, toys, linens,
mattresses and paints that are advertised as “antibacterial”. (David Suzuki Foundation) In between 2008
and 2009, annual consumption of TCS recorded 132 million liters. (Weatherly et al., 2017) Due to its
versatility, TCS is found in many environments, including surface water, wastewater, soil, drinking water,
wastewater treatment plants, landfills, and sediments. As TCS is commonly used in oral consumer
products, it is widely found in human urine, similarly to BPA. (Carey et al., 2015)
The European Union describes TCS as irritating to the skin and eyes, very toxic to aquatic organisms, and
with the potential to cause serious long-term effects in the aquatic environment due to its high solubility.
(David Suzuki Foundation) Environment Canada likewise categorises TCS as potentially toxic to aquatic
organisms (David Suzuki Foundation), bioaccumulative, and persistent, as it doesn’t degrade easily after
accumulation. TCS also reacts to form dioxins, which are toxic bioaccumulants (David Suzuki
Foundation). Approximately 1.1 × 105 to 4.2 × 105 kg of TCS are distributed to the environment annually
through WWTPs in the U.S alone. (Carey et al., 2015)
In 2016, TCS was banned by the FDA in soap products (liquid, gel, foam, bar); however, TCS still
remains allowed in other products such as toothpaste, hand sanitizer, and mouthwash, as TCS effects on
human and environmental health are still currently under debate. (Weatherly et al., 2017) Despite clear
correlation being yet to be confirmed, human TCS exposure through consumer products has been found to
be sufficient to produce adverse effects in cell types such as keratinocytes and oral mucosal cells that are
directly exposed to consumer products (Weatherly et al., 2017). ​At application concentrations TCS can
induce physical cell damage, causing cell contents to leak out through the membrane. (Carey et al., 2015)
Furthermore, TCS was shown to remain in the body for a half-life of 21 hours, meaning even brief use of
a TCS containing consumer product may result in a prolonged exposure period and thus a greater
likelihood for harmful effects in humans. (Weatherly et al., 2017)
Besides the health effects, TCS may also cause bacteria in the environment to be altered, become TCS
resistant, or resistant to other antimicrobial agents, following environmentally relevant exposure levels.
(Weatherly et al., 2017) Further, the extensive use of TCS in consumer products may contribute to the
proliferation of antibiotic-resistant bacteria. (12) At concentrations lower than 1 mg/L, TCS serves as an
external pressure to select for TCS resistance as well as antibiotic resistance in many types of bacteria.
(Carey et al., 2015) The mechanisms that convey resistance to TCS simultaneously cause resistance to
more than one class of antibiotics. (Carey et al., 2015)
7
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Figure 2: Lemna minor (Aquarium Breeder)
Lemna minor is known for its rapid growth and production of new offshoots, and its populations are an
important food source for aquatic organisms. This rapid growth rate has been beneficial for low-cost
scientific progress: for instance, the plants are being used for bioremediation of waterways that contain
excessive amounts of phosphorus and nitrogen from agricultural runoff. (US Forest Service) Researchers
are also developing techniques to use genetically modified duckweeds to synthesise insulin and other
commercially valuable proteins and new biomedicines. (US Forest Service) This research potential has
also been extended to its ability to degrade certain toxic substances from its environment. Furthermore,
Lemna minor has been studied for the effects of both BPA (Pop et al., 2021) and TCS (Boese). The
methodology in this paper is inspired by these studies, albeit for slightly different research purposes.
L.minor’s relevance as a food source and producer is also crucial for the purposes of this investigation, in
demonstrating the threats posed to the wider freshwater ecosystem if such key members of a food chain
were to be exposed to endocrine disruptors, even assuming that trophic transfer does not occur.
Figure 3: Artemia NYOS (Olsen)
8
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Sea-Monkeys (artemia NYOS) are a hybrid breed of brine shrimp (artemia salina), which shares many
similarities to the wild species. (Harvey, 2022) Artemia salina live in high salinity environments, such as
brine pools (dubbing them the common name brine shrimp). Both species display leaflike limbs, which
they beat to move. (Harvey, 2022) Artemia salina eggs can last for several years when freeze dried; this
simulates a unique biological process - known as "cryptobiosis”- wherein adverse environmental
conditions cause them to enter suspended animation and stay in that state indefinitely, until conditions
improve. (Harvey, 2022) Artemia NYOS feed on a diet of yeast and spirulina, and are translucent,
breathing through their feathery feet; they reproduce both sexually or asexually, and they chase flashlight
beams. (Harvey, 2022)
Such fascinating qualities aside, the reason behind the selection of artemia NYOS lies in the more
practical field: it involves no special equipment to maintain its populations (ie. they involve low costs),
are highly adaptable to a wide salinity range and temperatures from 6 to 35°C. (Therezinha et al., 2018)
They have a short life cycle and high fecundity, and are adaptable for many nutrients as they are
non-selective filterers. (Therezinha et al., 2018) There is also a lack of ethical implications, as brine
shrimp lack a developed nervous system which would allow them to feel pain. Thus, artemia are ideal for
both short and long term toxicological studies, allowing relatively less rigorous methodology,
effectiveness, and a high cost benefit ratio. (Therezinha et al., 2018)
The brine shrimp lethality test is a general bioassay used for the preliminary assessment of cytotoxicity in
dental materials, marine natural products, antitumor agents, pesticides, and plant extracts for
pharmacological activity. (Oladipupo et al., 2013) Such reports have shown a very positive correlation
between the lethality of substances to brine shrimp and antitumoral activity in the development of new
anticancer drugs from plants. (Oladipupo et al., 2013) The methodology for this investigation was
inspired by such previous studies, but more specifically one that records the cytotoxic activities of select
plant-derived essential oils (Waghulde et al., 2019). The acute toxicity test with brine shrimp (Artemia
sp.) is designed to expose a known number of Artemia larvae during 24 to 48 hours to the target chemical
substance in an aqueous saline sample. After this time, the number of dead organisms is quantified to
determine the lethal toxic effect. (Therezinha et al., 2018)
Similar to lemna minor, they are a significant low trophic food source in aquatic (marine) environments,
meaning that results may successfully convey the damage that can be imposed onto the wider marine
ecosystem if such key members of a food chain were to be exposed to endocrine disruptors, even
assuming that trophic transfer does not occur.
Methodology 1: Solution Making
1.1: BPA Solutions
Equipment
●
●
4 x 300-350 ml Glass beakers
4 x Small evaporating dish
Procedure
1. Label 4 sterile beakers: “200 ppm BPA”,
“150 ppm BPA”, “100 ppm BPA”,
9
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
●
●
●
●
●
●
●
●
●
●
Plastic funnel x 1
Labelling Tape
Cling Film
10 g of Bisphenol A (BPA) granules
Narrow metal spatula x 1
Glass stirring rod x 1
20 ml of ethanol (%100)
Micropipette (100 μl -1000 μl) x 1
Micropipette tip
1.5 L distilled water
“50ppm BPA”.
2. Measure precisely 0.06g (200ppm),
0.045g (150ppm), 0.03g (100ppm), and
0.015g (50ppm) of BPA granules and
place in separate evaporating dishes
3. Carefully add 4000μl of ethanol into each
of the 4 evaporating dishes.
4. Stir the contents of each evaporating dish
gently using a sterile glass rod.
5. Move the contents of each evaporating
dish into the 4 separate beakers, using a
clean funnel to prevent spillage.
6. Add 296 ml of distilled water to each
beaker. This should produce 4 beakers in
total, each with 300ml solutions of
different BPA concentrations.
1.2: TCS Solutions
Equipment
●
●
●
●
●
●
●
●
●
●
●
●
●
4 x 2L Plastic Containers
5 x 300-350 ml Glass beakers
1 x small evaporating dish
Plastic funnel x 1
Labelling Tape
Cling Film
0.001g of Triclosan (TCS) powder
Narrow metal spatula x 1
Glass stirring rod x 1
5ml of ethanol (%100)
Micropipette (100 μl -1000 μl) x 1
Micropipette tip
4 L distilled water
Procedure
1. Make 1000 ppb TCS: Prepare 2000μl of ethanol in an evaporating dish. Using a narrow metal
spatula, add precisely 0.001g of TCS powder. Stir at a moderate speed with a sterile glass stirring
rod. Once fully dissolved, transfer the mixture into a sterile container (A), preferably using a
clean plastic funnel to prevent spillage.
10
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
2. Add 998 ml of distilled water to the same container, and stir thoroughly. Take aside 300 ml of this
final mixture for use. Store in a sterile glass beaker and cover the opening with cling film - label:
“1000 ppb TCS”.
3. Add 700ml of distilled water to the remaining mixture in the container (A). This results in 1400ml
of a 500ppb TCS solution. Take aside 300 ml for use and store in a sterile glass beaker, covering
the opening with cling film - label: “500 ppb TCS”.
4. Take out another 200 ml of the remaining mixture in container (A) and pour into a different
container (B).
5. Add 800 ml of distilled water to the mixture in container (B), forming 1000ml of 100ppb TCS
solution. Take aside 300ml for use and store in a sterile glass beaker, covering the opening with
cling film - label: “100 ppb TCS”.
6. Take out 500 ml of the remaining mixture in container (B) and pour into a different container (C).
7. Add 500 ml of distilled water to the mixture in container (C), forming 1000ml of 50 ppb TCS
solution. Take aside 300ml for use and store in a sterile glass beaker, covering the opening with
cling film - label: “50 ppb TCS”
8. Take out 200ml of the remaining mixture in container (C) and pour into a different container (D).
9. Add 800 ml of distilled water to the mixture in container (D), forming 1000 ml of 10 ppb TCS
solution. Take aside 300ml for use and store in a sterile glass beaker, covering the opening with
cling film - label: “10 ppb TCS”.
10. In the end, there should be 5 beakers containing 300ml each of the 5 different concentrations of
TCS solution.
11. Empty any remaining mixture from containers (A),(B),(C),and (D) into the chemical waste bin
(or any other appropriate method that prevents contamination of public water supplies).
Investigation 1 : EDC and Duckweed
Organism care
For optimal growth conditions pH values were maintained at between 6.5 and 8, while the temperature
was regulated at 22 degrees celsius. (Michael, 2021) (iNaturalist NZ) While it is necessary for lemna
minor to be supplied with an additional application of nitrogen, phosphorus and potassium when grown in
rainwater, this was foregone to reduce the number of factors that may affect the plant besides the
concentration of BPA and TCS.
Methodology 2 : EDC and Duckweed
Equipment
●
●
BPA: solutions prepared at 200 ppm, 150
ppm, 100 ppm, 50ppm
TCS: solutions prepared at 10ppb, 50ppb,
100ppb, 500ppb, 1000ppb
Variables
Independent
Concentration of EDC solutions; BPA [ 50 ppm,
100 ppm, 150 ppm, 200 ppm]
and TCS [ 10 ppb, 50 ppb, 100 ppb, 500 ppb,
1000 ppb]
11
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
●
●
●
80 ml Glass Beakers x 27 (3 per
concentration for both BPA and TCS)
Duckweed (lemna minor)
Cling film
Dependent
The growth of l.minor clusters and buds,
measured as percentage change from initial
numbers.
Controlled
Number of initial clusters: set to 20 across all
concentrations and trials.
Light exposure: all beakers containing the l.minor
were placed in the same location with the same
amount of light availability throughout the day.
Carbon dioxide availability: the atmospheric
environment was the same for all l.minor in this
investigation.
Water available: the volume of solution was
maintained at 80ml throughout the investigation,
to ensure water availability was consistent.
Procedure
1. Place 80 ml of solution in each beaker. Label accordingly.
2. Carefully lift and place 20 buds of l.minor in each beaker
3. Cover each of the beakers with cling wrap to avoid contamination of solution
4. Poke holes in the cling wrap to allow gas exchange for photosynthesis.
5. Count the number of clumps and buds (separately) on 24 hour intervals.
6. Record the results on a data table: over 7 days for BPA solutions and 8 days for TCS solutions.
7. The average rate of l.minor budding: calculate daily growth rate for 7 days (7 intervals) at all 4
concentrations and divide the values obtained by the number of days.
8. Repeat for each trial and then average once again through dividing the summed values by 3.
9. The overall percentage change in the number of buds: count the change in the number of buds in
between day 1 and day 7. Convert into a percentage of the number on day 0.
10. Repeat for all 4 concentrations, and all three trials. Take the average by dividing values obtained
by 3.
BPA Results Data
Qualitative data
Throughout the 7 day exposure period to BPA, lemna minor individuals were seen to obtain a darker
disposition, and by the end of the investigation most buds displayed a near black hue.
12
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Raw data
Table 2.1: Trial 1 counting of l.minor clusters over 7 days
Concentration of BPA
Trial 1
0 ppm (ctrl)
50 ppm
100ppm
150 ppm
200 ppm
D0
20
20
20
20
20
D1
25
25
30
23
22
D2
26
29
37
23
22
D3
26
29
35
23
22
D4
28
30
35
23
22
D5
29
30
35
23
22
D6
29
31
35
23
22
D7
28
31
35
23
22
Processed Data
Table 2.2: Change (%) over 7 days in the number of clusters in l.minor exposed to BPA
Conc (BPA) (ppm)
50
Average Growth (%change in clusters over 7 days)
61.7
100
150
200
55
6.7
10
Graph 1: Percentage Change in the number of l.minor clusters over 7 days of BPA exposure
13
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Analysis
While there is a general decrease in the development rate of new clusters as the concentration of BPA
increases, the change in number of l.minor clusters demonstrates no coherent trend. It may be worthwhile
to note that the growth of clusters at 150 ppm was similar to that of the higher concentration 200 ppm, or
in fact more severely stunted. These results suggest that an increase in the concentration of BPA leads to
stunted growth in l.minor, but not necessarily in a linear manner.
Raw data
Table 3.1: Trial 1 counting of l.minor buds over 7 days
Concentration of BPA
Day #
0 ppm (ctrl)
50 ppm
100ppm
150 ppm
200 ppm
D0
20
20
20
20
20
D1
52
43
41
38
22
D2
61
58
55
49
40
D3
61
59
59
49
40
D4
65
59
61
49
46
D5
69
61
61
50
47
D6
71
61
64
51
47
D7
75
66
68
52
49
Processed data
Table 3.2: Trial 1 Percentage Change in the number of buds, across 2 day intervals (+overall change)
Concentration of BPA
Interval
0 ppm (ctrl)
50 ppm
100 ppm
150 ppm
200 ppm
D0-D1
160
115
105
90
10
D1-D2
17.3
58.537
34.146
28.947
81.818
D2-D3
0
1.7241
7.2727
0
0
D3-D4
6.5573
0
3.3898
0
15
D4-D5
6.1538
3.3898
0
2.0408
2.1739
D5-D6
2.8986
0
4.918
2
0
14
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
D6-D7
5.6338
8.1967
6.25
1.9608
4.2553
Average
28.363
26.693
22.997
17.564
16.178
D0-D7 (total)
275
230
240
160
145
Table 3.3: Average rate of budding (%) across 3 trials in l.minor over the 7 day exposure period (BPA)
Conc (BPA)
50 ppm
100 ppm
150 ppm
200 ppm
% change
24.993
19.958
15.498
12.978
Graph 2: Average rate (%) of l.minor budding over 7 days of BPA exposure
Table 4: Overall change (%) in the number of buds (BPA)
​
Conc (BPA)
50ppm
100ppm
150ppm
200ppm
% change
230
206
136
115
Graph 3: Overall change (%) in the number of buds (BPA)
15
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Analysis
Similarly to the trend in cluster proliferation, the number of buds decreases as the concentration of BPA
increases. There is a consistent and steady decline, in both the average rate of budding and overall
percentage change (growth). This again supports the negative correlation between increasing BPA
concentrations and growth in l.minor.
TCS Results Data
Qualitative data
Throughout the 7 day exposure period to TCS, lemna minor individuals were seen to obtain a lighter
disposition, and by the end of the investigation a majority of the buds displayed a near white hue.
Figure 5: lemna minor, Day 1 (left) and Day 3 (right)
Raw data
Table 5.1: Trial 1 Change in the number of clusters in l.minor exposed to BPA (remaining data in
appendix)
Concentration of TCS
Day #
0 ppb (ctrl)
10 ppb
50 ppb
100 ppb
500 ppb
1000 ppb
D0
20
20
20
20
20
20
D1
26
21
20
21
20
20
16
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
D2
27
21
21
22
21
22
D3
26
22
21
22
22
22
D4
27
22
22
23
22
22
D5
27
23
23
23
22
23
D6
27
26
23
23
22
23
D7
27
26
23
23
22
23
D8
28
26
23
23
22
23
%change
40
30
15
15
10
15
Processed data
Table 5.2: Percentage Change over 7 days in the number of l.minor clusters (TCS)
Conc (TCS)
10 ppb
50 ppb
100 ppb
500 ppb
1000 ppb
Average Growth (% change in clusters over 7 days)
43.3
20
20
23.3
13.3
Graph 4: Percentage Change in the number of l.minor clusters over 7 days of TCS exposure
Analysis
These results again show a general decrease in the development of new clusters as the concentration of
TCS increases. However, as the average growth of clusters is greater at 500 ppb than at lower
concentrations such as 50 ppb and 100 ppb, this demonstrates a non-linear negative correlation between
increasing concentrations of the endocrine disruptor and the growth of l.minor. Furthermore, the results
produced at concentrations 50 ppb and 100 ppb are equal at 20% change. This suggests that the inhibitory
effect of TCS increases drastically at concentrations between 10 ppb and 50 ppb, and less so for
concentrations of 50 ppb and up.
17
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Raw data
Table 6.1: Trial 1 counting of l.minor buds over 7 days
Concentration of TCS
Day #
0 ppb (ctrl)
10 ppb
50 ppb
100 ppb
500 ppb
1000 ppb
D0
43
48
46
39
35
40
D1
52
51
49
41
36
40
D2
53
51
50
47
43
41
D3
62
52
58
50
49
42
D4
69
54
59
51
50
45
D5
77
56
75
53
51
47
D6
81
61
79
54
51
49
D7
84
61
83
57
52
49
D8
84
64
84
58
52
49
Processed data
Table 6.2: Trial 1 Percentage Change in the number of buds, across 2 day intervals (+overall change)
Concentration of TCS
Interval
0 ppb (ctrl)
10 ppb
50 ppb
100 ppb
500 ppb
1000 ppb
D0-D1
20.93
6.25
6.5217
5.1282
2.8571
0
D1-D2
1.9231
0
22.449
14.634
19.444
2.5
D2-D3
16.981
1.9608
2.0408
6.3829
13.953
2.439
D3-D4
11.29
3.8462
1.7241
2
2.0408
7.1429
D4-D5
11.594
3.7037
27.119
3.9216
2
4.4444
D5-D6
5.1948
8.9286
5.3333
1.8868
0
4.2553
D6-D7
3.7037
0
5.0633
5.5556
1.9608
0
D7-D8
0
4.918
1.2048
1.7544
0
0
Average
8.9521
3.701
8.9319
5.1579
5.282
2.5977
D1-D7 (total)
95.349
14.583
82.609
48.718
42.857
22.5
18
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Table 6.3: Average rate (%) of l.minor budding over the 7 day exposure period (TCS)
Conc (TCS)
10 ppb
50 ppb
100 ppb
500 ppb
1000 ppb
% change
6.49
7.55
4.97
5.76
2.18
Graph 5: Average rate (%) of l.minor budding over 7 days of TCS exposure
Table 7: Overall change (%) in the number of buds in l.minor exposed to TCS
Conc (TCS)
10 ppb
50 ppb
100 ppb
500 ppb
1000 ppb
% change
48.47
71.51
93.16
48.85
17.5
Graph 6: Overall change (%) in the number of buds (TCS)
Analysis
The average rate of l.minor budding was measured through the same process as in table sets 6 and 7. The
highest average rate of budding occurs at a concentration of 50 ppb, and the lowest average rate occurs as expected - in the highest concentration of 1000 ppb. The greatest overall change in the number of buds
occurs in 100 ppb, and the least change in 1000 ppb. There is no significant trend in the budding rate or
19
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
overall growth among the values obtained for concentrations greater than 0 ppb. For instance, the rate of
budding and overall growth in 10 ppb are lower than those in 50 ppb. This indicates that TCS has had a
greater disruptive effect on the growth of l.minor at the lower concentration of 10 ppb. Meanwhile, 50
ppb produces lower overall growth than in 100 ppb (this implies that it has greater disruptive effects than
the solution with double the concentration). Similarly, 500 ppb TCS produces a greater average rate of
budding than 100 ppb (ie. it has a weaker inhibitory effect on budding than lower concentrations) but
lower overall budding (similar levels to 10 ppb). These results demonstrate non-linearity in both the
average rate of budding and overall budding in l.minor individuals exposed to TCS of a range of
concentrations.
Investigation 2: EDC and Sea Monkeys
Organism care
The artemia NYOS were incubated in glass beakers, in an environment created according to the sea
monkey care manual. (The Original SeaMonkeys). Oxygen was pumped daily using an aquatic air pump
and sandstone. Temperature was 25 degrees for hatching and then kept at 22 degrees celsius during
growth to reduce the impact of fluctuating temperatures on the life of sea monkeys.
Methodology 3: EDC and sea monkeys
Equipment
●
●
●
●
●
BPA: solutions prepared at 200 ppm, 150
ppm, 100 ppm, 50ppm
TCS: solutions prepared at 10ppb, 50ppb,
100ppb, 500ppb, 1000ppb
Sea monkeys (artemia NYOS)
27 x 300-350 ml Glass beaker (3 per
concentration for both BPA and TCS)
Cling film
Variables
Independent
Concentration of EDC solutions; BPA [ 50 ppm,
100 ppm, 150 ppm, 200 ppm]
and TCS [ 10 ppb, 50 ppb, 100 ppb, 500 ppb,
1000 ppb]
Dependent
The mortality rate of artemia NYOS; calculated
from the number of dead individuals throughout
11 days of exposure.
Controlled
Oxygen availability: oxygen levels were
maintained through the use of an aquatic air
pump, twice a day for 60 seconds each.
Salinity: Salinity of the original hatching
environment was maintained through the addition
of distilled water after periods of evaporation.
Nutrition: artemia NYOS individuals were given
the spirulina and yeast mixture provided by the
Sea Monkey care kit.
20
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Temperature: monitored with a thermometer and
maintained at 22 degrees celsius
Procedure
1. Ensure that there are 10 sea monkeys well and present in each beaker.
2. Ensure that there is 300 ml of liquid in each beaker holding the sea monkeys.
3. Add 10 ml of solution into the beaker; label as you proceed. For example, if 10 ml of 200 ppm
BPA solution is added to the beaker, label it “200 ppm BPA”.
4. Place and firmly secure cling film to the opening of each beaker; this will prevent the
contamination of the trial.
5. Poke 10 small holes in the cling film to ensure oxygen is available.
6. Make observations and record mortality data in 24h intervals for 10 days.
7. At the end of each interval, the number of dead individuals are counted and the percentage of
mortality is determined using the following equation:
% mortality = (no. of dead individuals/initial no. of live individuals) x 100
BPA Results Data
Qualitative data
The artemia NYOS individuals exposed to BPA progressively developed relatively slow pace motion,
within 2 or 3 days of the investigation. The bodies of dead specimens were completely immobile and
gradually developed a darker hue.
Figure 6: Immobile artemia NYOS, prior to discoloration
Raw data
Table 7.1: Trial 1 Number of dead artemia NYOS individuals across 11 days (BPA)
Concentration of BPA
Hours 50 ppm
100 ppm
150 ppm
200 ppm
54
0
3
4
1
21
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
65
7
0
6
6
72
7
7
8
6
90
8
10
8
6
96
8
10
8
6
117
8
10
9
8
168
8
10
9
8
265
8
10
9
8
Processed data
Table 7.2: Average Mortality rate across 3 Trials (BPA)
Concentration of BPA
Hours
50 ppm
100 ppm
150 ppm
200 ppm
54
7.037
0
27.777
43.333
65
55.926
3.3333
58.89
55
72
55.926
60
79.63
58.333
90
62.963
83.333
79.63
68.333
96
79.63
100
89.63
81.666
117
86.296
100
93.33
100
168
86.296
100
93.33
100
265
86.296
100
100
100
Graph 7: Average artemia NYOS mortality rates across 265 hours of BPA exposure
22
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Analysis
As depicted in the graphs above, 100% mortality rate is reached before the end of 11 days, although only
in the 3 highest concentrations. This demonstrates a relatively linear relationship between the
concentration of BPA and its mortal effect on organisms. Despite this general trend, the rate of increase in
mortality through the whole 11 days demonstrates a weaker correlation with the concentration of BPA.
For instance, 100 ppm BPA had the slowest initial effect on mortality; up to the 65 hour mark, this and the
control specimen are the only two that have not affected any artemia NYOS individuals. However, 100
ppm BPA reached 100% mortality in the shortest period of time (96 hours).
200 ppm BPA led to the quickest initial effect on mortality (which aligns with its potency as the highest
concentration), but was the second slowest in reaching 100% mortality (117 hours). As expected, the
lowest final mortality rate was produced by the lowest concentration of 50 ppm; this was the only solution
(besides the control) that did not reach 100% mortality rate.
TCS Results Data
Qualitative data
Artemia NYOS individuals developed and maintained swift regular motion throughout the investigation.
Post-mortem individuals were completely immobile and displayed a discoloured and translucent physical
state.
Figure 7: Immobile Artemia NYOS, following discoloration.
Raw data
Table 8.1: Trial 1 Number of dead artemia NYOS individuals since initial exposure (0h)
Concentration of TCS
Hours
10 ppb
50 ppb
100 ppb
500 ppb
1000 ppb
54
0
1
2
1
0
65
1
1
2
2
0
23
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
72
1
1
4
3
0
90
1
1
4
6
0
96
1
1
4
7
0
117
2
1
4
7
2
168
2
1
4
7
3
265
2
1
4
8
3
Processed data
Table 8.2: Average Mortality Rate of artemia NYOS (% change since 0 hours)
Concentration of TCS
Hours
10 ppb
50 ppb
100 ppb
500 ppb
1000 ppb
54h
0
12.5
25
10
0
65h
12.5
12.5
25
20
0
72h
12.5
12.5
50
30
0
90h
12.5
12.5
50
60
0
96h
12.5
12.5
50
70
0
117h
25
12.5
50
70
25
168h
25
12.5
50
70
37.5
265h
25
12.5
50
80
37.5
Graph 8: Average artemia NYOS mortality rates across 265 hours of TCS exposure
24
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Analysis
As depicted in the graphs above, none of the concentrations of TCS resulted in a 100% mortality rate. Alll
mortality rates recorded from the TCS trials were lower than those of the BPA trials. This may suggest the
relative impact of each substance, but does not provide any ground for comparison in the long term as the
duration of the experiment was only 11 days. The highest final mortality rate was in 500 ppb TCS, at
80%. There is a relevant positive and linear trend in the final rates of mortality as the concentration
increases, with the exception of 1000 ppb. 1000 ppb had the slowest initial effect on mortality; up to the
117 hour mark, no artemia NYOS individuals were affected. Furthermore, 1000 ppb resulted in only the
third highest final mortality rate: this may indicate that for TCS, lower concentrations may have a greater
disruptive effect on the life of sea monkeys. For instance, 100 ppb led to the quickest initial effect on
mortality: however, it is shown that the mortality rate does not change from 50% after the 72 hour mark,
implying that the overall impact lasts for a shorter period of time. Furthermore, the lowest final mortality
rate is found in 50 ppb, not the lowest concentration (10 ppb). Once again, this suggests that TCS effects
do not correlate linearly with the concentration of solutions.
Discussion
The results of this investigation may be explained through scientific context: for instance, research
specific to the bioaccumulation of BPA in aquatic species suggests that the EDC does not remain in those
organisms for long via aquatic exposure, due to the nature of their metabolism. (Corrales, 2015) In a study
involving rainbow trout (Corrales, 2015) it was shown that BPA is readily absorbed into the liver, plasma,
and muscle of the specimen; each of the compartments reached maximum (100%) BPA concentrations 2
hours after injection. However, 24 hours following injection, only 1.5%, 2.0%, and 1.7% BPA remained
in the liver, plasma, and muscle, respectively. Similarly, inhalational exposure of BPA through water
displayed a relatively short (<6 hours) half-life in fish plasma and tissues. This rapid elimination of BPA
in fish is likely due to the nature of their metabolism; it was observed that rainbow trout and zebrafish
rapidly convert BPA to other less toxic substances such as BPA glucuronic acid and BPA sulphate, which
are then primarily excreted in bile through the intestine. (Corrales, 2015)
This may to an extent suggest an explanation for the length of time it took for the EDCs to take effect in
the sea monkeys. In the brine shrimp lethality assay (Waghulde et al., 2019) that was referred to for parts
of the methodology in this investigation, the substances were tested for 24 hours, with 100% mortality
rates being observed in this time frame. Contrastingly, in both the trials for BPA and TCS, none of the sea
monkeys were dead by the 24 hour mark, and the earliest time any were found dead was 54 hours after the
initial exposure. While this may imply that the duckweed and sea monkey are able to metabolise and
excrete the EDCs from their systems to an extent, such results may also be attributed to the substances
being tested. The original method involved plant derived essential oils, which are certainly different from
BPA and TCS: EDCs are suspected to be more hazardous in the long run. Note that these investigations,
while longer than 24 hours, were carried out in a relatively short period of time and may not accurately
convey trends in long term exposure. In addition, the size of the organisms tested (l.minor and artemia
NYOS) may have led to higher mortality rates in a short period of time (relative to larger organisms).
25
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
Nonetheless, the results of this investigation show that the substances can be highly toxic for organisms in
lower trophic levels. While it produces no directly relevant data on the bioaccumulation of the EDCs, it
demonstrates mortality at all ranges of concentrations. As both l.minor and artemia NYOS are located in
the lower levels of the food chain, this implies the potential for disastrous effects if trophic transfer is
possible and active. Even foregoing trophic transfer, these results suggest potential fluctuations in
population numbers of producers and small aquatic organisms if EDCs were to contaminate the waters in
larger concentrations.
Another factor to consider are design errors. While laboratory records of BPA bioaccumulation are fairly
low, field bioaccumulation factors for BPA in the same organisms investigated are typically much higher.
(Corrales, 2015) This suggests that laboratory experiments may not accurately simulate the complex
conditions of EDC exposure in aquatic habitats and organisms; a factor which may have affected the
results of this investigation. For instance, other factors such as nutrient composition and salinity in the
solutions may have affected the growth and death of l.minor and artemia NYOS respectively. Moreover,
artemia NYOS’ genetically modified nature may cause disparities between real life effects of EDC in
aquatic ecosystems and those observed in this investigation.
Conclusion
In agreement with the hypothesis, the results demonstrated a negative correlation between increasing
concentrations of BPA and growth in lemna minor (specifically the budding of duckweed). In agreement
with the hypothesis, there was a strong positive correlation between increasing concentrations of BPA and
mortality in artemia NYOS. While there was a general agreement with the hypothesis in that the
development of clusters in lemna minor was stunted with increasing concentrations of TCS, there was no
linear or exponential correlation found. While there was a general agreement with the hypothesis in that
the final mortality rates for artemia NYOS increased with increasing concentrations of TCS, lower
mortality rates at the highest concentrations demonstrated the lack of a linear correlation. Therefore,
contrary to the hypothesis, results demonstrated the lack of consistent correlation between increasing
concentrations of TCS and the growth of lemna minor or the mortality in artemia NYOS.
While there seems to be no literature values that support or negate the results of this investigation, they
corroborate with the concept of complex dose-effect relationships in EDCs. (Lauretta, 2019) Unlike other
toxic substances, the increase in the concentrations of EDCs will not result in a directly proportional
increase in impact or range of their toxic effect. As seen in this investigation, different concentrations of
both BPA and TCS led to diverging effects - in terms of the time window of impact. This supports that the
variety in dosage produces a non-traditional trend in response, which could be explained in context of
how EDCs function - the complex nature of processes involving biochemical signalling, hormones, and
their receptors. (Lauretta, 2019)
These results are also significant in terms of the societal implications of highly water-soluble EDCs such
as BPA and TCS. As demonstrated in the results, both substances had a negative effect (both in the short
term and long term) on the life and development of aquatic organisms. Although concentrations of EDCs
present in humans are suspected to be much lower than those used in these investigations, the potential of
bioaccumulation and bioamplification in non-human animals ( Lauretta, 2019 ), as well as the potential
26
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
for trophic transfer, strongly suggest the magnitude of harm that could result from rising levels of these
toxic substances in our aquatic environments.
Therefore, relevant areas for further research may be methods for the removal of EDCs from our land and
waters. A few examples that are currently under study include the biodegradation of BPA utilising
lignolytic enzymes sourced from fungi, non-pathogenic bacterial strains, and aquatic organisms such as
duckweed.
Appendix
1. Trial setup for Investigation 1 : EDC and Duckweed
2. Additional raw data for Investigation 1 : EDC and Duckweed - BPA exposure
Concentration of BPA
Trial 2
0 ppm (ctrl)
50 ppm
100 ppm
150 ppm
200 ppm
D0
20
20
20
20
20
D1
46
43
31
36
24
D2
59
49
52
42
33
D3
60
49
59
43
36
D4
65
50
63
43
38
D5
71
50
63
43
38
27
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
D6
77
52
63
44
38
D7
83
53
64
44
40
Trial 3
0 ppm (ctrl)
50 ppm
100 ppm
150 ppm
200 ppm
D0
20
20
20
20
20
D1
60
50
32
27
30
D2
69
67
39
43
38
D3
71
74
49
44
38
D4
76
75
49
44
38
D5
88
76
51
44
38
D6
89
76
52
46
39
D7
92
79
52
46
40
3. Additional raw data for Investigation 1 : EDC and Duckweed- TCS exposure
Concentration of TCS
Trial 2
0 ppb (ctrl)
10 ppb
50 ppb
100 ppb
500 ppb
1000 ppb
D0
42
51
48
45
31
48
D1
51
55
53
47
34
49
D2
57
61
55
53
43
50
D3
63
77
60
54
48
50
D4
72
84
65
59
48
50
D5
85
84
73
60
48
50
D6
101
87
74
62
49
50
D7
110
96
74
65
49
54
D8
118
102
77
65
49
54
Trial 3
0 ppb (ctrl)
10 ppb
50 ppb
100 ppb
500 ppb
1000ppb
D0
20
20
20
20
20
20
D1
39
51
58
44
32
30
D2
53
55
50
49
50
42
28
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
D3
65
68
72
51
60
52
D4
82
70
65
52
60
47
D5
88
76
70
54
60
53
D6
107
82
81
57
60
53
D7
111
85
88
57
65
54
D8
113
88
80
56
64
50
4. Additional raw data for Investigation 2: EDC and Sea Monkeys - BPA exposure
Concentration of BPA
Trial 2
0 ppm (ctrl)
50 ppm
100 ppm
150 ppm
200 ppm
54h
0
1
0
2
4
65h
0
5
0
5
4
72h
0
5
6
7
5
90h
0
6
8
7
6
96h
0
8
10
9
8
117h
0
9
10
9
10
168h
0
9
10
9
10
265h
0
9
10
10
10
Trial 3
0 ppm (ctrl)
50 ppm
100 ppm
150 ppm
200 ppm
54h
0
0
0
3
4
65h
0
4
1
6
5
72h
0
4
5
8
5
90h
0
4
7
8
7
96h
0
7
10
9
9
117h
0
8
10
9
10
168h
0
8
10
9
10
265h
0
8
10
10
10
29
The Effect of Endocrine Disruptors on the mortality rate of Lemna minor (Duckweed) and Artemia NYOS (Sea Monkeys)
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