REVIEW OF LITERATURE

Nature of Bioassay

A bioassay is a procedure that uses living organisms to determine the toxicity

of a chemical. (Cahill, 2006) It is one method of assessing the presence of potentially

harmful compounds. (http://pulse.pharmacy.arizona.edu)

In a bioassay, living organisms are exposed to different concentrations of a

chemical that might be toxicant (harmful chemical). Observations of the effects on the

organisms’ behavior and survival allow researchers to determine if, or at what

concentration, a chemical has harmful effects. Typically researchers test the effects of

toxicants on model organisms that are small and easy to maintain in a laboratory

setting. (Cahill, 2006)

For environmental testing, bioassays provide an integrated picture of overall

toxicity of an effluent or a sample of water, sediment, or soil from a contaminated

site. Fathead minnows, various aquatic invertebrates, earthworms, protozoan, and

seed all are used for bioassay of aquatic samples. The idea behind this bioassay is that

the test organism will react in a predictable way to various types of environmental

contaminants. Several studies have compared the sensitivities of various types of

seeds to common pollutants. (Keddy, 1995)

Toxicity testing has grown steadily in recent years. It is an important

parameter in wastewater quality monitoring as it provides the complete response of

test organisms to all compounds in wastewater. They are useful because they integrate

the toxicity of all factors associated with the medium such as interactive effects

between chemicals. For example, chemical A might be toxic at a high concentration,

but if in the presence of chemical B, chemical A becomes toxic at a much lower

concentration. Conversely, chemical C might be toxic at a low concentration, but in

the presence of chemical D, chemical C becomes much less toxic and requires much

greater concentrations to cause an effect. In addition, different organisms are sensitive

to different chemical concentrations and mixtures. (EIM, 2013)

Water Pollution in Urban Areas

Urban areas have the potential to pollute water in many ways. Run-off from

streets carries oil, rubber, heavy metals, and other contaminants from automobiles.

Untreated or poorly treated sewage can be low in dissolved oxygen and high in

pollutants such as fecal coli form bacteria, nitrates, phosphorus, chemicals, and other

bacteria. Treated sewage can still be high in nitrates. Groundwater and surface water

can be contaminated from many sources such as garbage dumps, toxic waste and

chemical storage and use areas, leaking fuel storage tanks, and intentional dumping of

hazardous substances. Pollution can lead to acid rain, nitrate deposition, and

ammonium deposition, which can alter the water chemistry of lakes. (Living Lakes

Partnership)

Uncontrolled or treated run-off from the urban environment and from

construction activities can run-off the landscape into surface waters. This runoff can

include such pollutants as sediments, pathogens, fertilizers/nutrients, hydrocarbons,

and metals. Pavement and compacted areas, roofs, and reduced tree canopy and open

space increase runoff volumes that rapidly flow into our waters. This increase in

volume and velocity of runoff often causes stream bank erosion, channel incision and

sediment deposition in stream channels. In addition, runoff from these developed

areas can increase stream temperatures that along with the increase in flow rate and

pollutant loads negatively affect water quality and aquatic life.

Other common sources of urban pollution include improperly sited, designed

and maintained onsite wastewater treatment (septic) systems, pet wastes, lawn and

garden fertilizers and pesticides, household chemicals that are improperly disposed of,

automobile fluids, road deicing/anti-icing chemicals, and vehicle emissions.

(Environmental Protection Agency, 2005)

An international environmental group is again raising the alarm over pollution

in drinking water in the Philippines, as it recently discovered nitrate contamination of

several water sources in areas in Benguet and Bulacan(PhilSTAR.com, 2007 )

In its new report entitled “Nitrates in drinking water in the Philippines and

Thailand,” Greenpeace examined the nitrate levels in drinking water sources like

artesian wells and creeks in Benguet and Bulacan and their relation to nitrogen

fertilizer use in farming areas in the two provinces. (PhilSTAR.com, 2007 )

Greenpeace has been sending out warning signals that the quality of our

freshwater source is declining, and the study is yet another shocking example of how

water protection measures are inadequate. The study showed that five out of the 18

water sources where they took samples from, have nitrate contamination that are

“alarmingly well above the safety limits” of the World Health Organization, whose

standard is 50 mg/l of nitrates in drinking water. Communities think that the water

they drink every day is clean because physically, it doesn’t smell bad or look bad, but

it is actually laced with nitrates from fertilizers which people don’t normally associate

with pollution. (PhilSTAR.com, 2007 )

 Based on the study, the result of Greenpeace Water Patrol’s weeklong

investigation during the last week of September, drinking water from two artesian

wells in Buguias, Benguet, and three others in Angat, Bulacan were found to have

nitrate levels well above the WHO standard.The highest nitrate level was found in

groundwater in Buguias at 50 percent higher than the WHO standard, the study said.

Greenpeace warned that the pollution could have serious health implications for the

local population, especially since groundwater is the main source of their drinking

water. (PhilSTAR.com, 2007 )

The report’s author, Reyes Tirado, of the Greenpeace Science Unit in the

University of Exeter in the United Kingdom, said nitrate pollution of drinking water

from artesian wells correlates with intensive farming practices in the affected areas

where nitrogen fertilizers are applied in excess.

Nitrogen fertilizer consumption in Asia has grown dramatically, increasing

approximately 17-fold in the last 40 years. Fertilizer application rates are increasing

rapidly in some developing countries, reaching excess amounts that can no longer be

used by crops and which cause problems for human health and the

environment.Excess application of nitrogen fertilizers in intensive crop production

leads to nitrate pollution of the artesian wells that people use for drinking water.

(Tirado, 2007)

There’s really a problem on how the government makes or implements

policies against water pollution. The government is either not doing things properly or

doing it excessively. It’s about time that the government really looks into this and

takes concrete action to ensure non-contamination of clean water sources for the

people. (Baconguis, 2007)

Daphnia Magna as Test Organisms in Bioassays

The water flea Daphnia Magna is the most commonly used zooplankton in

toxicological tests in wastewater treatment, due to short doubling time, high

sensitivity, and simplicity; therefore, it was used as an indicator. (APHA, AWWA,

WEF, 1992; Official Gazette, 1996; USEPA, 2000)

This species is found in freshwater and brackish (up to 8 ppt salinity) habitats

including lakes, rivers, and temporary pools. Although they prefer temperatures

between 18-22°C, they can tolerate a much broader range. ( Elenbaas, M. 2013 as

cited in Ebert, 2005; Haney, 2010; Vanoverbeke, et al., 2007)

These water fleas are very small, usually 2-5 mm long, with an overall shape

similar to a kidney bean. The body is enclosed by a transparent shell-like structure,

called a carapace that is mostly made of chitin. Due to its transparent carapace, this

species tends to be the color of what it is currently eating. The carapace extends into

the head shields, an important diagnostic characteristic for this species. They have two

sets of long, doubly branched antennae and six thoracic appendages that are held

inside of the carapace and help to produce a current of water, carrying food and

oxygen to their mouths and gills. They also have two large claws, used mainly for

cleaning the carapace. They have one compound eye, which appears as an anterior

dark spot, and one simple eye (ocellus). Males are smaller than females (typically

only 2 mm long while females are 3-5 mm long) but have longer antennules and

modified, hook-like first appendages used for clasping females during mating.

(Elenbaas, M. 2013)

The life cycle begins when a female produces a clutch of eggs (usually 6-10)

that are released into her brood chamber, located under her carapace. Eggs hatch into
juveniles within this brood chamber and are released when their mother molts,

typically within 2-3 days. Juveniles, which already resemble adults, go through a

series of molts and instars. Females are considered sexually mature after developing

brood pouches, usually after 4-6 instars, usually 6-10 days. (Elenbaas, M. 2013)

Lifespan of these water fleas depends heavily on environmental conditions

such as oxygen levels, food availability, and temperature. In general, as temperature

decreases, lifespan increases, with averages of 40 days at 25°C and 56 days at 20°C.

Unstable environmental conditions tend to lead to shorter lifespans. While it has been

suggested that males of this species have shorter lifespans than females, recent

research shows evidence that this is likely not the case. (Clare, 2002; Grzesiuk, et al.,

2010; Pietrazak, et al., 2010)

Daphnia feed on small, suspended particles in the water. They are suspension

feeders (filter feeders). The food is gathered with the help of a filtering apparatus,

consisting of the phylopods, which are flattened leaf-like legs that produce water

current. As the current flows anterior to posterior, the Daphnia collect particles that

are transferred into the food groove by special setae. Although the feeding apparatus

is so efficient that even bacteria can be collected, the food is usually made up of plank

tonic algae. Green algae are among the best food, and most laboratory experiments

are done with either Scenedesmus or Chlamydomonas, both of which are easy to

culture in monoclonal chemo stats. Daphnia usually consume particles from around 1

μm up to 50 μm, although particles of up to 70 μm in diameter may be found in the

gut content of large individuals. (Ebert,2005)

The gut is more or less tubular with three parts: the esophagus, the midgut, and

the hindgut. There are two small digestive ceca(diverticula) that are easily seen in the
head section of the midgut. The midgut is lined with an epithelium and bears

microvilli. Peristaltic contractions of the gut wall pass food through the gut, but a

peritrophic membrane contains the food and prevents it from entering the ceca.

Epithelial cells do not phagocytose particles but absorb molecules. The pH is 6 to 6.8

in the anterior part of the midgut and 6.6 to 7.2 in the posterior part. Food is expelled

from the hindgut by peristaltic movement but also requires the pressure of more

recently acquired food particles. The color of Daphnia adapts to the food that is

predominant in their diet. Daphnia feeding on green algae will be transparent with a

tint of green or yellow, whereas those feeding on bacteria will be white or salmon-

pink. Well-fed animals are more strongly colored than starved animals. (Ebert,2005)

Daphnia have an open blood circulation. The heart is located dorsally and

anterior from the brood chamber. At 20ºC, it beats about 200 times per minute,

slowing down at lower temperatures. Blood cells are easily visible through the

transparent body as they flow rapidly through the body cavity. To support oxygen

transport, Daphnia have the extracellular respiratory protein hemoglobin (Hb), a

multi-subunit, multi-domain macromolecule. There are at least four Hb genes.

Daphnia tend to develop more Hb to increase oxygen uptake from the water. In

response to environmental changes (oxygen concentration, temperature), the Hb

concentration varies up to about 20-fold. Oxy-hemoglobin, the form that is loaded

with oxygen, is red and gives the transparent animals a reddish appearance. Because

certain parasites also cause the hem lymph to become red, one cannot easily

determine the cause of the red color from sight alone. However, low oxygen usually

affects an entire population, coloring all animals reddish, whereas parasites usually

infect only a portion of the population. (Ebert,2005)

Other Organisms Used in Bioassay

Duckweed it is a small aquatic plant that floats on the surface of ponds,

wetlands, nutrient rich lakes. Worldwide, there are over 40 species of duckweed

(Family Lemnaceae), with 20 species found in the United States. Each plant consists

of one or more fronds. The frond look like little leaves but actually rootlet that

dangles down in water. Although duckweed reproduces through budding- new fronds

grow from their roots and break off to become independent plant.

(http://ei.cornell.edu/toxicology/bioassays/Duckweed/)

Duckweed is useful for conducting bioassay experiments with water samples

because it can be measured by growth rate by counting how many new fronds develop

over a five-day period. By measuring the number of new fronds of duckweed plants

growing in a test solution and comparing that to the number of new fronds in control

solution, you can test the sensitivity of duckweed to different compounds, or various

concentrations of a single compound.

(http://ei.cornell.edu/toxicology/bioassays/Duckweed/)

Elodea is a rooted multi-branched perennial plant but can survive and grow as

floating fragments. The dark green blade-like leaves (3/5 inch long and 1/5 inch wide)

are in whorls of three with finely toothed margins. The flowers of Elodea have three

white petals with a waxy coating that makes them float.

(http://aquaplant.tamu.edu/plant-identification/alphabetical-index/elodea/)

Submerged portions of all aquatic plants provide habitats for many micro and

macro invertebrates. These invertebrates in turn are used as food by fish and other

wildlife species (e.g. amphibians, reptiles, ducks, etc.). After aquatic plants die, their

decomposition by bacteria and fungi provides food (called “detritus”) for many
aquatic invertebrates. Elodea has no known direct food value to wildlife but is used

extensively by insects and invertebrates.

Elodea is often confused with Hydrilla and Egeria. Elodea has only 3 leaves in

the whorl and no midrib teeth. (Department of Wildlife & Fisheries Sciences Texas

A&M AgriLife Extension Service)

Planarian is any of several free-living (non-parasitic) worms. The planarian is

the simplest of all flatworms. Most planarianare found in freshwater or salt water, but

a few live in moist soils on land.

The planarian has a soft, flat, wedge-shaped body that may be black, brown,

gray, or white and is about a half inch (1.3 cm) long. The blunt, triangular head has

two ocelli (eyespots), pigmented areas that are sensitive to light. There are two

auricles (earlike projections) at the base of the head, which are sensitive to touch and

the presence of certain chemicals. The mouth is located in the middle of the underside

of the body, which is covered with cilia (hair like projections). The nervous system

consists of a simple brain from which two nerve cords extend the length of the body.

Other nerves connect these cords, forming a ladder like structure. There are no

circulatory or respiratory systems; oxygen entering and carbon dioxide leaving the

planarian's body diffuses through the body wall.

The planarian travels with a gliding motion by moving its cilia. It can also

travel short distances by using a rapid crawling motion. The planarian feeds on

crustaceans, larvae, and small worms. To eat, it extends a long, tubular pharynx from

its mouth. Through this tube it secretes digestive juice onto its prey, and then sucks in

bits of partly-digested food.

 Planarian is hermaphroditic; that is, they possess both male and female sex

glands. However, a planarian cannot fertilize its own eggs; the eggs must be fertilized

by the sperm of another planarian. In another method of reproduction, a planarian

constricts behind the pharynx and separates itself into two pieces (a process called

fragmentation); then, both pieces grow back their missing parts (a process called

regeneration) and become two new planarians.

Scientists have used the planarian for studies on regeneration and for research

on learning and behavior. The planarian can learn to respond to a particular stimulus.

When such a planarian is cut into several pieces, the new planarians regenerated from

the pieces in many cases “remember” the learned response of the original planarian.

Planarian belongs to the class Turbellaria of the phylum Platyhelminthes.

There are many families and genera. (Source: Animal Planet)

Of all the possible water quality bioassay organisms, lettuce seeds might be

one of the last you would think of using. Lettuce seed bioassays have proven to be an

easy and inexpensive means of testing the toxicity of some types of contaminants of

concern in water and sediments, including heavy metals and some pesticides and

other organic toxicants.

Lettuce seeds provide distinct advantages over most other test organisms: they

are inexpensive, easy to culture, and require no upkeep between experiments.

Although any variety of lettuce might work, Lactucasativa L. var. Buttercrunch is the

standard species recommended for bioassays by the U.S. Environmental Protection

Agency, the Food and Drug Administration, and the Organization for Economic

Cooperation and Development.

 So for future studies, test organisms like duckweed, elodea , planaria and

lettuce seeds may be used for bioassay studies.

Other Researches on Using Daphnia Magna to Evaluate Toxicity

Several researches were also conducted using Daphnia magna as bio

indicator.

Villegas Navaro et al. (1999) reported the use of Daphnia Magna as a toxicity

indicator for textile industrial effluents to show that the toxicity tests combined with

physicochemical analysis are essential in the evaluation of effluent quality and also in

the assessment of treatment plant efficiency in Mexico.

The toxicity removal efficiency of different units of Istahan Wastewater

Treatment Plant (IWTP) was evaluated and the validity of regular physicochemical

parameters as limits for discharge to receiving waters. Daphnia was collected from a

natural park. Initially one of the isolated daphnia was cultured. In the next step, the

recultured daphnids are used to prepare the final culture. For this reason, 100 ml of

the final culture was poured into special bottles. Then, one single Daphnia was added

to each bottle. To support the growth of Daphnia during the day after initial culture

one mg of yeast was added to each bottle, every other day. Identification of Daphnia

was carried out according to US-EPA (2000). Thirteen samples were taken from four

different points of IWTP. Samples were taken from raw wastewater influent, and also

from preliminary, primary and secondary sedimentation tanks effluents. The samples

were diluted by 3, 4.5, 7, 15, 10, 23, 34, 51, 77, and 100% (v/v). Ten daphnids were

added to each dilution and the results of the daphnid mortality rate were recorded after

48 hours (LC50). The result of experiments was acceptable only in cases where

daphnids in the blank tubes were observed to have a mortality rate of less than 10%.
Totally, 520 samples were tested. It should be noted that temperature was checked

regularly using a thermometer in the culture medium. At the end of the experiment,

Acute Toxicity Unit (ATU), efficiency of each unit and totally efficiency values were

determined. (Movahedian, Bina and Asghari, 2005)

The result obtained for the influent to the effluent from each unit showing

48h-LC50 and ATU in the raw wastewater and in preliminary, primary, and secondary

treatment effluents. The results were analyzed using SPSS software and Probit

facility. The toxicity results obtained from IWTP shows 48h-LC50 for raw wastewater

as the effluent of the plant was 30% (v/v) and 3.3 as ATU (Acute Toxicity Unit). The

highest and lowest levels (95% confidence limit) were 48.5 and 21, respectively.

Similar results were reported by Blinova (2000) which reported 48h-LC50 up to 34%

(v/v) for raw wastewater. However, it should not be noted that the quality and

quantity of raw wastewater could be quite different due to culture, custom, nutrition,

health and education (Metcalf and Eddy, 2003). As the result of both studies were

found to be in the same range of 95% confidence limit, therefore, the accuracy of the

results is acceptable. The 48h-LC50 for wastewater effluent after preliminary

treatment was 32% (v/v) with the highest and lowest levels being 53 and 22,

respectively. The confidence limit was 95%. The ATU was found as 3.1. the LC 50

obtained in this step was not significantly compared with that of raw wastewater.

Preliminary treatment consisted of screening and grit removal. It was assumed that

some of the toxic materials, which were toxic to Daphnia, might be absorbed by grit

and large suspended solids and, thus, removed. However, the results of this study

showed that these units were not efficient in removing these materials. The toxicity

removal efficiency of preliminary treatment was found to be 8%.(Movahedian, Bina

and Asghari, 2005)

 An approach to compare the toxicities employing the whole effluent toxicity

(WET) test, using Daphnia magna and chemical analysis with GC/MS and ICP/MS,

was conducted to the nine South Korean wastewater treatment plants (WWTPs). From

the chemical analysis and bioassay experiments, heavy metals (i.e., Cu and Zn) were

found to be the major compounds causing toxic effects toward D. magna. In the whole

effluent toxicity (WET) tests using D. magna, toxicities were observed in 34% of the

effluent samples. However, the biological toxic unit (TU) value showed a non-toxic

response (i.e., 0 TU) in many samples despite the response indicated by the chemical

TU values. This may be due to the species sensitivity, environmental parameters,

mixture effects, and limitation of the chemical analyses.(Department of

Environmental Science and Engineering, Gwangju Institute of Science and

Technology, Buk-Gu, Gwangju, South Korea, 2008 )

Whole effluent toxicity (WET) tests, with Daphnia magna and Selenastrum

capricornutum, were introduced to evaluate the biological toxicities of effluents from

the wastewater treatment plants (WWTPs) in Korea. In WET tests of WWTPs

effluents, 33.3% (33/99) for D. magna and 92.6% (75/81) for S. capricornutum

revealed greater than 1 toxic unit (TU), even though all the treatment plants

investigated were operating in compliance with the regulations, as assessed using

conventional monitoring methods (i.e., BOD and total concentration of N or P, etc).

There were only minor differences in toxicities according to the types of influents

(municipal and agro-industrial) in all treatment plants. However, the effluents treated

by an activated sludge treatment process were found to exhibit significantly lower

toxicity than those treated by rotating biological contactor (RBC) and extended

aeration processes. The seasonal variations in the toxicity were lower in the summer

compared to winter, which may have been due to the rainfall received to the sewage
intake system during the former period. The impact of WET on river water was also

investigated based on the discharge volume. At sites A and B, the total impact of

toxicity to stream and river waters was observed to be 70.9% and 90.4% for D. magna

and S. capricornutum, respectively. The other four small treatment plants (sites F, G,

H and I), with relative discharging volumes between 0.001 and 0.002, contribute less

than 1% to the total toxicity. (Environmental Monitoring & Assessment;Jun2007,

Vol. 129 Issue 1-3, p107)

Another study entitled, “Evaluation of water treatment sludges toxicity using

the Daphnia bioassay” was conducted. Alum and ferric chloride sludges from two

water treatment plants (WTPs) were analyzed regarding their physicochemical

characteristics and toxicity to Daphnia similis. Experiments were carried out in the

dry and rainy seasons. Acute and chronic toxicity was measured using survival and

reproduction as measurement endpoints. No acute toxicity of the sludge was observed

in 48 h exposure. Ferric chloride sludge caused chronic toxicity, demonstrated by low

fecundity and some mortality, while alum sludge caused chronic toxicity

characterized by low fecundity. Some sludge characteristics varied between

samplings, including turbidity, solids contents, N, P and metal (Al and Fe)

concentrations. These variables and the increase of chemical oxygen demand (COD)

were identified as the main cause of degradation of the receiving waters. However, no

relationship was observed between these variables and degree of toxicity. It is

apparent from these results that water treatment sludges may be toxic and therefore

may impair receiving waters. Alum sludge was less toxic than ferric chloride sludge.

In their study entitled,” Toxicity assessment of a complex industrial wastewater using

aquatic and terrestrial bioassays Daphnia pulex and Lactuca sativa,” aquatic and
terrestrial bioassays were used to assess toxicity at several stages in an industrial

wastewater treatment plant that processes 400 L/s from a complex influent formed by

wastewater from 135 industries. Daphnia pulex and Lactuca sativa were used to

assess and compare toxicity between the influent wastewater and effluent wastewater

from an activated sludge process, and compare their relationship with

physicochemical parameters of Biological Oxygen Demand (BOD); Chemical

Oxygen Demand (COD); Total Suspended Solids (TSS); total Nitrogen (N (N-total)),

and ammonia Nitrogen (N (N - NH3)). Samples from the primary clarifiers (PC), mix

liquor stage (ML) and secondary clarifiers (SC) were processed using

physicochemical and bioassay test. Toxicity results with Daphnia pulex showed

decreased mean values of acute Toxic Units (a.T.U.) between PC (2.1 a.T.U.) and SC

(1,25 a.T.U.). Lactuca sativa showed high values of toxicity between PC and SC (3.37

and 3.32 a.T.U. respectively). Some samples exhibited higher toxicity values at the

effluent stage (SC) than the influent stage (PC). The highest correlations of

physicochemical properties with toxicity were obtained with COD and nitrogen

compounds in effluent samples (SC), but not with influent samples (PC). (Sánchez-

Meza JC, 2007)