Whole Effluent Toxicity Testing

Dr. Akepati S. Reddy

Dept. Biotechnol. Environ. Sci.
Thapar University
 What causes toxicity?
One or more of
• Chemical factors:
– Inorganic chemicals: ammonia, chlorine, heavy metals,
etc.
– Organic chemicals: dioxins, polychlorinated biphenyls
and surfactants
– Pesticides: chlorpyrifos, diazinon and heptachlor, etc.
• Physical factors:
– Dissolved and suspended solids
– Temperature
• Biological factors:
– Bacteria, fungi, and parasitic invertebrates
 Whole Effluent Toxicity (WET) test

First methods for conducting toxicity tests were

published in around 1945
Standard prescribed in India (Bioassay test)
‘90% survival of fish after 96 hours in 100% effluent’
(Schedule-VI and Schedule-I of
EP Rules, 1986 under the EPA, 1986)
Objective behind the standard: to satisfy the ‘no toxics
in toxic amounts’ in the receiving waters
 Important terms
LC50 (Lethal Concentration): Concentration (in %) at which
the effluent is lethal to 50% of the exposed test organisms
at the specified time of observation
NOAEC (No Observed Adverse Effect Concentration): lowest
concentration of effluent (in %) at which the test
organisms’ survival is not significantly different from the
control
TUa (Acute Toxic Units): 100/LC50
IC25 (Inhibition Concentration): concentration of effluent (in %) at
which inhibitory effect on the test organisms is 25% when
compared when compared with that of the control – chronic test
related
NOEC (No Observable Effect Concentration): Highest effluent
concentration (in %) at which no statistically significant effect on
the organisms is there when compared with the control –
chronic test related
 Whole Effluent Toxicity Test
A parameter for measuring aggregate toxic effect of
effluents discharged into surface waters
Important component of an integrated approach for
detecting and addressing toxicity in surface waters
Allows determination of impacts of exposure of aquatic
life to effluent without knowing its characteristics
– Assessment & accounting for of chemical interactions
– Addressing of unknown toxicants
– Assessment of bio-availability of toxic constituents
Limitations of the WET test
– Fails to address carcinogenicity, mutagenicity and
bioaccumulation phenonmena
– Fails to cover persistence of toxicants on sediments
– Allows incomplete knowledge of causative toxicants
 Whole Effluent Toxicity Test
WET involves answering ‘Is the sample toxic to the test
species? If yes, how toxic is it to the test species?’
By WET one can know if particular characteristics of
receiving water are rendering the effluent with toxic
chemicals non-toxic
– receiving stream water should be used as dilution
water
If methods and species are standardized comparison of
WET results become possible
In WET test species are exposed to undiluted and
diluted effluent under controlled conditions for varying
periods for assessing toxicity and its extent through
measuring survival, growth and/or reproduction of the
test organisms
 Whole Effluent Toxicity Test
Includes two types of tests: Acute and Chronic
• Acute toxicity tests
– Exposure time is shorter (96 hours or less)
– Focus is on the survival of the test organisms (Mortality
is considered as end point)
– Indicates only lethal concentrations
– Only fast acting chemicals can exhibit their effect
– These can be static non-renewal tests, static renewal
tests or flow through tests
– Less expensive and end point is easily quantifiable
• Chronic toxicity tests
– Exposure is for longer period relative the organisms life
cycle usually during very sensitive life stage
– Focus is on survival, growth and/or reproduction
 Test method
Test frequency, test species, test method and reporting
of results are as prescribed
Test method used is influenced by
– Exposure regime to be tested
– Nature of the receiving water body
– Resident species of the stream
– Variety of other factors
Test species is placed in the sample in a series of
concentrations (100, 50, 25, 12.5, 6.25% and control)
– Observations are made at specific time intervals
– Test duration may be 40 min to 7 days depending on
the organisms used and on the type of effects (acture
or chronic) interested to known
 Test method [IS 6582 (part-2): 2001]
Range finding test
– To find the effluent concentration range within which 0
to 100% mortality is observed within 24 hours
– Use of serial dilutions of effluent sample is
recommended
– Effluent volume is 2 L
Standard test (96 hour duration)
– Dilution within the range on geometric progression
scale is recommended
– 100%, 50%, 25%, 12.5%, 6.25% and so on
– Use of five consecutive dilutions and one control is
recommended
– Effluent volume is 2 L
– Performing test with one replication is recommended
Test conditions [IS 6582 (part-2): 2001]
• Feeding of fish should be stopped 24 hours before
• All fish for a test should come from the same stock
• 5 randomly picked up fish per vessel should be used
• Fish should not be harmed while transferring (transferring
should be completed within 30 min.)
• Samples and test solutions should not be treated/aerated
• Count dead fish after 2, 6, 12, 24, 48 and 96 hrs.
• If any fish die in the control vessel discard the test and start
fresh test
• Measure DO, pH and temperature in each vessel after 24
hr and 48 hours
• Carry out the tests under normal laboratory illumination
conditions with natural photoperiod
• Maintain temperature at 25±1°C
 Collection, handling and preservation
of sample
Sampling location: same as that for other parameters
Sample type: Grab or Composite
– Grab samples offer snap shot of the effluent while composite
samples offer average view of the effluent
– Compositing is for 24 hours and sampling interval is 0.5 hr
Material of sample container: Borosilate glass, Teflon like
perflurocarbons, 304 or 316 grade stainless steel, or
plastics like plyethylene, polystyrene, polypropylene and
polycarbonate
Rinse container with effluent prior to sample collection
Sample can be held for <24 hrs and only temperature
preservation (refrigeration or icing) of sample is allowed
 Dilution water
Standard laboratory water or receiving water can be used
- choice depends on the objectives of the test
– Standard laboratory water is preferred for absolute
toxicity
– Receiving water body is good for estimating toxic effect
on receiving water body
– Use receiving water if it is uncontaminated otherwise
use standard laboratory water
Standard laboratory water [IS 6582 (part-2): 2001]
• 25 ml/L each of the following to water
– 11.76 g/L of CaCl2.2H2O in water
– 4.93 g/L of MgSO4.7H2O in water
– 2.59 g/L of NaHCO3 in water
– 0.23 g/L of KCl in water
• Aerate dilution water for 24 hours prior to use in the test
 Test species
Brachydanio rerio (Hamilton Buchanan) of size 30±5 mm and
weight 0.2 to 0.3 g [IS 6582 (part-2): 2001]
Sensitive organisms which are easily cultured and readily
available year round
– response should be consistent and reproducible
Both plants and animals can be used - fish, invertebrates and
algae - Species used depend on
– Characteristics of receiving water (salinity) – fresh water
or salt water
– Applicable toxicity standards
– Whether the interest is in acute or chronic effects
Organisms used are cultured under carefully controlled
laboratory conditions
– In some cases test organisms collected from field sites
are used for WET testing
 Stocking of fish
• At 25°C in aerated chlorine free potable water of
characteristics roughly matching with dilution water
• Population density of 1g/L
• Daily illumination range of 12 to 16 hours
• Diet is normal diet
• 10 days minimal acclimatisation period
• Mortality should not be >1%
 Whole Effluent Toxicity Test
WET tests give information for
– for evaluating impacts of effluent on the survival, growth,
reproductive capacity and normal development of test
populations
– For the estimation of concentration above which detrimental
impacts from the effluent can occur in receiving stream
Statistical approaches are used to analyze the WET test
data include
– Comparing the results of test dilution with non-toxic
reference water to identify significant reductions in an
organisms’ response in the test dilution
– Mathematically model relationship between sample
concentration and response of test organism and use
the model to identify the concentration that causes
specific level of response
 Whole Effluent Toxicity Test
• Result are compared with the toxicity standards
• If consistent toxicity is observed in samples from the
same source over time, Toxicity Information
Evaluation (TIE) procedures are used to identify the
effluent characteristics causing the toxicity
• Once toxicity causing characteristics are identified,
Toxicity Reduction Evaluation (TRE) procedures are
used to outline and evaluate effective approaches to
reduce the effluent toxicity
 References for more information
• Methods for Measuring the Acute Toxicity of Effluents to
Freshwater and Marine Organisms. 5th Edition, USEPA,
Office of Water, October 2002, EPA 821-R-02-012
• Short-term Methods for Estimating the Chronic Toxicity of
Effluents and Receiving Waters to Freshwater Organisms.
4th Edition, USEPA, Office of Water, October 2002,
October 2002, EPA 821-R-02-013
 Test method
Temperature, light, DO, pH, salinity, conductivity,
residual chlorine total hardness, alkalinity and
chamber size and volume are specified
DO should not fall below 4 mg/l
Test organism feeding is another organism should
be standardized
Test acceptability criteria are fixed
Sampling technique is specified
Test measurements are fixed
 Test method
Test frequency, test species, test method and reporting
of results are as prescribed
Test method used is influenced by
– Exposure regime to be tested
– Nature of the receiving water body
– Resident species of the stream
– Variety of other factors
Test species is placed in the sample in a series of
concentrations (100, 50, 25, 12.5, 6.25% and control)
– Observations are made at specific time intervals
– Test duration may be 40 min to 7 days depending on
the organisms used and on the type of effects (acture
or chronic) interested to known