Haqal Aufarassya Anwar

2006576823
TL - 01
TUGAS INDIVIDU
RINGKASAB BAB 2
HANDBOOK OF INDUSTRIAL AND HAZARDOUS WASTES TREATMENT

Chapter two will discuss the importance of assessing risks caused by contaminants in the

environment to both human health and ecosystems. The ecological risk assessment or (ERA) is a key

tool for estimating the effects of chemical or physical sensors within the environment. Toxicity

bioassays are crucial evidence in ERA, the demand for sensitive bioassays using native organisms to

detect pollution has been increasing. Aquatic ecotoxicology and integrated biological/chemical

strategies must be developed further to understand the impact of chemical agents on biological integrity.

Bioassays are proven to be alternatives to measure environmentally relevant toxicity compared to

chemical analysis. Bioassays are cost effective, precise, and sensitive in detecting adverse

environmental conditions. The main drawback of biological effect measurement is to find the link

between observed effects to specific pollution aspect. In certain situations, biological analysis cannot

completely replace chemical analysis but can complement it in some situation.

There are multiple key considerations related to ERA and toxicity tests. These main points

include:

1. Problem Formulation in ERA : The problem formulation phase involves identifying the

components at risk of an ecosystem and specifying the endpoints used to assess and

measure that risk. Where Assessment endpoints represent valued resources, while

endpoints are the actual data measures for evaluation.

2. Classification of Toxicity Tests : Toxicity tests can be categorized based on several aspects

which are: exposure time, specific responses, or mode of effect. Acute toxicity tests are an

example of short-term tests focusing on lethality expressed as LC50 or EC50 values, while

chronic toxicity tests assesses long-term effects, including sublethal ecological impacts and

involve the full life-cycle or early-stage tests.

 3. Toxicity Measurement Parameters : Multiple parameters are used such as LC50, EC50,

NOEC, and LOEC in order to measure toxicity.

4. Use of mesocosm studies : Mesocosm studies provide a more comprehensive view of the

ecological impact of toxic substances since they involve a larger and more diverse

ecosystem. These studies are often conducted outdoors

5. Selecting relevant test organisms : Tests organisms should and must align with the

assessment endpoints, represent the functional roles in ecosystems and be sensitive to

contaminants.

6. Applications of Biological Toxicity Tests : These tests are often used to evaluate toxicants

in liquid waste such as in wastewater treatment plants, process control, and toxicity

reduction evaluations.

7. Ecotoxicological testing for solid wastes : Necessary for multiple aspects such as risk

assessment, assessing contaminant extractability affecting groundwater, evaluation of

waste for future use as soil amendments, etc.

8. Elutriate Toxicity Tests vs Contact Toxicity Tests : There are two approach for solid waste

toxicity tests and each approach has their own advantages and disadvantages. Water

elutriation has a chance to underestimate bioavailable organic contaminants, while contact

tests will consider interactions with solid-phase substances

Microbial toxicity tests are known for being fast, simple and inexpensive, resulting in an

increase of popularity for environmental control and assessing pollutants in waste. They are also

valuable for classifying the toxicity samples from the same origin. These tests are also capable of

involving pure cultures of a single species or a mixture of various microbe species. Microbial tests are

also capable of measuring various variables including lethality, growth rate, changes in species

diversity, decreased degradation, etc. The outcomes of these toxicity tests are most commonly

expressed in values such as EC50 or EC10. There are various toxicity testing methods within the

environmental assessment and waste management which includes:

 1. Tests based on Bioluminescence : Using Vibrio Fischeri bacteria to measure light emission

changes when exposed to toxins, quick and reliable method to assess toxicity in water,

wastewater, etc. Results are expressed as the inhibition of light emission.

2. Tests based on enzyme activity : Measure changes in enzyme activity caused by

contaminants. One example is the ECHA Biocide Monitor as a simple, field-applicable tests

that is capable of evaluating dehydrogenase activity.

3. Tests based on growth inhibition : Using Pseudomonas putida to measure toxicity by

monitoring bacterial growth. This test is mostly useful for assessing sewage sludge, soil

extracts, and chemicals.

4. Tests based on the inhibition of Motility : This test makes use of Spirillum volutans to

quickly screen wastewater or sample quality.

5. Tests based on respiration measurements : Using methods such as respiration inhibition

kinetics analysis (RIKA) to study parameters such as qmax and KS allowing the assessment

of toxic compound effects. The Polytox kit may also be used to measure respiratory activity

changes in bacterial cultures when exposed to contaminants.

6. Genotoxicity : Typically assesses mutagenic potential using Salmonella bacteria, other tests

may use different bacteria such as E.Coli. These tests evaluate the DNA-damaging

properties of waste contaminants

7. Tests based on nutrient cycling : Provides insights into how contaminants and pollutants

affect nutrient cycle and processes of microorganisms within the environment.

Fauna toxicity tests may be conducted with as an approach with different taxon, there are

multiple groups of fauna that are used for toxicity testing among many are crustaceans, protozoa,

cnidaria, fish, and invertebrates. Sub chapter 2.4 will discuss the differences between toxicity testing of

each group .

1. Crustacean testing : Particularly the Daphnia species of crustaceans, are considered

essential for ecotoxicological tests. Daphnia are recommended by international

organizations such as US EPA, EEC, and OECD. These Daphnia tests are even required for

regulatory testing in many countries, many factors attribute to the popularity of Daphnia
 testing which are : they are common in freshwater environments, have a short life-cycle,

easy to cultivate in labs, are sensitive to various contaminants, require minimal space and

resources. Within acute and chronic bioassays Daphnia magna and D.pulex are commonly

used. As mentioned before, these tests are highly recommended as they are standardized,

cost-effective, and reproducible.

2. Protozoa testing : Protozoa has multiple species that can act as indicators in the presence

of toxic contaminants. Examples such as Colpodium campylum’s growth inhibition may be

measured in presence of toxicants. Other protozoa such as Paramecium caudatum are

suitable for wastewater toxicity.

3. Cnidaria testing : Known for ease of handling, and observable morphological changes in

response to toxicants. Cnidaria such as the Hydra attenuata are often used in assessing

acute lethal toxicity in wastewaters.

4. Fish testing : Traditionally, fish would be used in industrial wastewater toxicity assessment,

but due to recent concerns of costs and ethics, researches are seeking alternatives in the

form of in vitro cell systems among many others.

5. Invertebrate testing : Invertebrate are used often used to evaluate the effects of toxic

contaminants. Examples include earthworms, collembola, and enchytraeid. Various species

are used to measure endpoints such as mortality, reproduction, and behaviour to asses the

toxic contaminants mentioned.

Algae testing are also used as test organisms in toxicity testing alongside microbial and fauna.

Organizations such as APHA, ASTM, ISO, and OECD provide standard algal toxicity test methods.

Selenastrum capricornutum and Scenedesmus subspicatus are the two commonly employed algal

speciaes for algae testing. Recent research are focusing on simplifying and reducing costs for algal tests,

these research has resulted in innovative methods such as the microplate techniques and algaltoxkit F.

Where the microplate technique is a method where algae are exposed to toxins in 96-well microplates

for 96 hours, within this method cell density is measured using a haemocytometer . The main benefits

of this method include smaller sample volumes, reduced space requirements, more replicated,

automation potential among many others. The microplate technique is a miniaturized version of the
conventional flask method. The algaltoxkit is an alternative method in algal testing. S. capricornutum

are used within testing and do not require pretest culturing. Growth inhibition in the presence of toxic

contaminants are measured with optical density in “long cell” test cuvettes.

Growth inhibition becomes the main parameter in algal toxicity testing, where an assessment

of cell concentration for each sample concentration over time (typically 24, 48 and 72 hours) is done

resulting in a growth curve. The EC50 is calculated at each time point using the area underneath the

growth curve to estimate the effect on biomass. The area given under the growth curve for each time

point is calculated using a formula. Meanwhile percent growth inhibition for each concentration is

obtained by comparing the area of the control growth curve with that of the concentration of the sample

being tested. Regression analysis is used to establish the EC50 and the concentration-effect curves.

Plant testing becomes has an interesting basis since plants are significant components in an

ecosystem due to their role in capturing solar energy while also producing essential resources such as

oxygen and sugars. Plants are sensitive to environmental chemicals, resulting in plants and their

usefulness as bioindicators. Plant tests within environmental analysis typically fall into five categories

being : biotransformation where tests are conducted to detect chemical changes caused by plants, food

chain uptake to determine the entry of toxic chemicals into food chains from plant absorption,

phytotoxicity as an assessment of toxicity and hazard pollutants pose to plant growth and longevity,

sentinel as a form of monitoring pollutants by observing symptoms in plans, and surrogate to replace

animal or human assays with plant-based tests.

Specifically phytotoxicity tests, focusing the impact of toxic contaminants on vegetation often

receive significant attention. A variety of plant species and endpoints such as seedling growth, seed

germination, and root elongation are used to characterize the effects of toxic contaminants. These plant

tests are often conducted in pots or petri dishes. OECD provides guidelines for plant toxicity testing

alongside a 100 plant taxa list in ASTM. This assessment involves the monitoring of plant growth within

a 14 day period and calculating the reduction in growth compared to control plants. Another parameter

for phytotoxicity assessment is the emergence of seeds within the plants. Other tests such as plant

growth and germination tests are often used to determine compost maturity and toxicity. Phytotoxicity

parameters help assess the potential for agricultural use or soil rehabilitation of different waste types.
The composting process is commonly used to reduce phytotoxic substances before use in soil

rehabilitation.

Table 1. Plant Species Recommended for Assessment of Toxicity by OECD

Sumber: (Wang and Hung 2004)

Other forms of bioassays that are available include Toxkit tests, in which are short-term

bioassays that assess the impact of foreign substances on aquatic environments and wastewaters. These

tests are rising in popularity due to their practicality compared to other tests. They are capable of

eliminating the need of continuous recruitment and culturing of live test species, which are often

expensive and requires skilled personnel. Other advantages included being efficient and cost effective

for toxicity screening, another big advantage lies within the microtox test, where the test kit uses marine

bacteria and is applicable anywhere without the need for culturing. Another innovative test is the “cyst-

based” toxicity tests that use dormant stages of cysts of aquatic invertebrate species. These cysts are

easily stored for extended periods and hatched when there is demand, ensuring a continuous supply of

test organisms. Commercial products for measuring toxicity are readily available, simplifying the

assessment of toxic contaminants and their impacts on aquatic environments.

 Subchapter 2.8 describes and explores the many uses of bioassays in assessing the ecological

risk of toxic contaminants within solid waste. Single species toxicity tests such as the microtox toxicity

test are considered an initial step in ecological risk assessment both soil and water. For instance, with

microtox toxicity test we are able to estimate the amount of copper toxicity found within sewage sludge

especially when combined with the microtox solid-phase method. There has been research that

discovered how copper toxicity in sewage sludge may increase significantly within the presence of

certain organic substances. These are the cases that highlight the importance of considering proper

organic compounds when assessing the suitability of sludge for soil amendment. Other various

applications of single species bioassays are assessing complex industrial wastes, process water,

wastewater effluents, sediment extracts, among many others.

Table 2. Commercially Available Toxicity Tests

 Sumber : (Wang and Hung 2004)

Genotoxicity also plays a crucial role in waste assessment, and tests like the Ames test using

Salmonella, SOS-ChromotestTM, and MutatoxTM are commonly used for studying genotoxicity within

waste, contaminated soil, sewage sludge, and sediments. In other studies, a battery of toxicity tests is

used to evaluate solid waste comprehensively. These tests include among many bioluminescent bacteria

tests, root elongation tests and germination tests. The results produced from these tests help determine

whether composted waste products are suitable for the environment and cause minimal or zero negative

environmental impacts.

Ecotoxicological procedures have been adopted by several countries in order to classify and

manage waste based on both chemical and biological analyses. These methods are used in order to

detect and manage hazardous waste properly and effectively. For instance, Russia uses a combination

of both chemical and biological analysis to categorize sewage sludge into different hazard classes. Other

countries namely Hungary and the Czech Republic also use environmental bioassays in order to

evaluate and classify wastes based on its toxicity. All in all, it is proven that bioassays play a significant

role in assessing ecological risk and sustainability of solid waste, helping ensure the safe management

of waste materials.

Subchapter 2.9 delves into the historical development and evolution of using bioassays in order

to meet regulatory requirements concerning effluents. Toxicity roots back to the ancient times,

Aristotle’s early experiments with aquatic organisms exposed to different environmental conditions was
one of the first examples noting toxicity. Formal efforts to control and assess water pollution through

testing began in the 1940s with attempts to standardize these tests around the 1950s. As noted effluent

in the book several times effluent toxicity testing serves multiple key purposes in pollution control such

as preventing and reducing effects in receiving water bodies, conducting compliance testing as part of

permit formulation, and guiding improvements in effluent quality through technological advancements.

In order to use effluent toxicity data effectively for pollution control, a proper and representative effluent

sampling is critical, considering the potential variations in quantity and toxicity. Effluent toxicity test

species are often different from resident species within an ecosystem, and not all species would respond

to toxic contaminants uniformly. Hence it is recommended to use multiple toxicity test organisms to

assess effluent toxicity comprehensively. There are drawbacks and limitations of single-species tests

such as overlooking species interactions, using genetically homogenous test populations, alongside the

fact that testing species may not accurately represent the resident species within natural ecosystems. As

mentioned, selecting a diverse test species from different trophic levels is essential to achieve ecological

realism in testing.

Multiple countries have a system of control and monitoring of industrial effluents. There are

limitations of solely relying on quantitative chemical analysis for control, while also emphasizing and

prioritizing the need for ecotoxicological testing in order to assess the real hazard of effluent discharges.

Among those countries, Canada, USA and Germany have managed to adopt systems to evaluate the

environmental hazards of industrial effluents, this is achieved by incorporating a combination of toxicity

tests. Another country namely Japan has also recognized the importance of ecotoxicological monitoring

but has not fully implement or endorse it. France, the United Kingdom, and Ireland often use biological

testing for toxicity tests, meanwhile Poland, Estonia, Hungary, the Czech Republic, Norway, and

Lithuania have fully developed standardized test programs for permit derivation, applying multiple

toxicity tests. Russia employs a diverse battery of tests for taxation purposes, hence revealing the

toxicological load on receiving water bodies. Each country has their own methods and approach in

controlling and monitoring industrial effluents, emphasizing the importance of ecotoxicological

bioassays within environmental management.

 It must be noted that there is a growing interest in utilizing biological tests such as bioassays as

a valuable tool in the risk assessment of waste materials, namely in liquid waste environments. There

is a variety of toxicological bioassays available and ready to use, yet only few have been adopted for

routine toxicological evaluations. This is caused mostly by the challenges in maintaining long-term

cultures of test organisms and also the lack of standardization. Using bioassays should be context-

dependant, considering multiple factors such as screening, regulatory requirements, or predictive hazard

assessment. Toxicity tests provided by bioassays offer a valuable means to assess the impact of complex

contaminant mixtures, while it must be reminded that bioassays also have their limitations. There is an

emphasis on the use of bioassays in conjunction with other appropriate tools within a risk assessment

framework, play an essential role in identifying, characterising, and mitigating the toxic effects of

contaminants from waste discharges on ecosystems.