ASTM E1768-95(2013)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: E1768 − 95 (Reapproved 2013)
Standard Guide for
Ventilatory Behavioral Toxicology Testing of Freshwater
Fish
This standard is issued under the fixed designation E1768; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope signals, such as those elicited in ventilatory behavior, in saline
(>2 ppt) or high conductivity (>3000 µmhos/cm) water using
1.1 This guide covers information on methods to measure
the procedures discussed in this guide. Therefore, this guide is
and interpret ventilatory behavioral responses of freshwater
restricted to the testing of freshwater matrices.
fish to contaminants.
1.8 The values stated in SI units are to be regarded as
1.2 Ventilatory responses are often some of the first prele-
standard. No other units of measurement are included in this
thal symptoms exhibited by animals to environmental stressors
2 standard.
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Continued, abnormal ventilatory
1.9 This standard does not purport to address all of the
behavior (that is, rapid or shallow breathing, erratic breathing)
safety concerns, if any, associated with its use. It is the
can indicate physiological damage that may be irreversible.
responsibility of the user of this standard to establish appro-
Such damage could eventually result in decreased survival,
priate safety and health practices and determine the applica-
growth, or reproduction of the organism, or all of these.
bility of regulatory limitations prior to use. For specific safety
1.3 Ventilatory responses of some fish species can be
precautions, see Section 6.
measured relatively easily and quickly, providing a useful tool
1.10 This guide is arranged as follows:
for biomonitoring studies of wastewaters, pure chemicals,
Section
surface water, and ground water.
Number
Scope 1
1.4 Appropriate studies of ventilatory responses can yield
Referenced Documents 2
definitive endpoints such as no observable effect concentration
Terminology 3
(NOEC) or an EC , often more rapidly than standard toxicity
Summary of Guide 4
Significance and Use 5
test methods (11, 12).
Safety Precautions 6
1.5 The mode of action of test substances and the type of Responses Measured 7
Test System 8
chemical toxicant can be determined by examining ventilatory
Test Procedure 9
behavioral responses in conjunction with other physiological
Data Collection and Analysis 10
responses (8, 9, 10, 11, 12). Interferences 11
Documentation 12
1.6 Fish ventilatory behavior can be assessed in real-time
References 13
using appropriate computer hardware and software (12, 13, 14,
2. Referenced Documents
15, 16, 17, 18, 19). Such systems have proved useful for
long-term, on-line monitoring of wastewater effluents, pure 3
2.1 ASTM Standards:
chemicals, and surface waters (12, 15, 20, 21, 22, 23, 24, 25).
E729 Guide for Conducting Acute Toxicity Tests on Test
These systems are usually technically complex and will not be
Materials with Fishes, Macroinvertebrates, and Amphib-
discussed in this guide.
ians
1.7 Given the technological constraints of electrical E943 Terminology Relating to Biological Effects and Envi-
ronmental Fate
components, it is currently not feasible to monitor bioelectric
E1192 Guide for ConductingAcute Toxicity Tests onAque-
ous Ambient Samples and Effluents with Fishes,
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental Macroinvertebrates, and Amphibians
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved March 1, 2013. Published March 2013. Originally
approved in 1995. Last previous edition approved in 2008 as E1768 – 08. DOI: For referenced ASTM standards, visit the ASTM website, www.astm.org, or
10.1520/E1768-95R13. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
The boldface numbers given in parentheses refer to a list of references at the Standards volume information, refer to the standard’s Document Summary page on
end of the text. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1768 − 95 (2013)
E1241 GuideforConductingEarlyLife-StageToxicityTests software. With the aid of a computer and analog to digital
with Fishes board, responses can be monitored and analyzed on a real-time
E1604 Guide for Behavioral Testing in Aquatic Toxicology basis. The computer-analyzed response reduces potential sub-
jective biases due to manual analysis of strip-chart recordings.
3. Terminology
5. Significance and Use
3.1 The words “must,” “ should,” “ may,” “can,” and
“might”haveveryspecificmeanings.“Must”isusedtoexpress
5.1 Responses that reflect oxygen consumption or utiliza-
an absolute requirement, that is, to state that the test ought to tion have often been targeted as useful indicators of incipient
be designed to satisfy the specified condition, unless the
toxic conditions (26, 27, 28, 29, 30). In addition, sustained
purpose of the test requires a different design. “Must” is only acute fish ventilatory behavioral responses reflect a physiologi-
used in connection with the factors that directly relate to the
calchangeintheorganismandthereforemighthaveecological
acceptability of the test. “ Should” is used to state that the relevance.
specified condition is recommended and ought to be met if
5.2 For some time, the technological means have been
possible. Although a violation of one “should” is rarely a
available to log and display ventilatory signals over time.As a
serious matter, violation of several will often render the results
result, there are a considerable number of studies which
questionable. Terms such as “is desirable,” “is often
examined ventilatory behavior of fish and other aquatic organ-
desirable,” and “might be desirable” are used in connection
isms. A large number of substances at lethal levels have been
with less important factors. “ May” is used to mean “is (are)
shown to elicit ventilatory responses relatively quickly (13, 19,
allowed to,” “can” is used to mean“ is (are) able to,” and “
20, 31, 32, 33, 34). For many pollutants, a significant response
might” is used to mean “could possibly.” Thus the classic
was often generated in less than1hof exposure to concentra-
distinction between “ may” and “can” is preserved, and
tions approaching the 96 h LC50. Studies performed using
“might” is never used as a synonym for either “may”or“ can.”
subacutely toxic samples of effluents or individual pollutants
3.2 Definitions of Terms Specific to This Standard: (concentrations well below the reported LC50 concentration),
3.2.1 cough—gill purge in fish; when a fish reverses or
often documented responses within 1 to 10 h of exposure (11,
greatly increases the flow of water over the gills and back out 18, 21, 30, 35, 36).
to the ambient water. Such activity is used to cleanse the gills
5.3 Given the data obtained thus far, it appears that fish
by removing particles or other material on the gill plate(s).
ventilatory behavior may be a very sensitive and rapid indica-
3.2.2 electrode—device (metallic or chemical based) that
tor of acute toxicity if various aspects of this behavior (that is,
receives bioelectric signals from the organism.
rate and amplitude) are assessed and analyzed simultaneously.
It appears that the more aspects of ventilatory behavior that are
3.2.3 ventilation—breathing, respiratory process of organ-
assessed,themoresensitiveandrapidthesystemis (11, 12, 21,
ism.
22).
3.2.4 waveform—representation of analog electrical signal
5.4 Although a variety of organisms have been examined
depicting breathing response of organism over time, usually
represented on a strip chart recorder or computer monitor. including crayfish (37), aquatic insect larvae (31), and bivalves
(13), most research in aquatic ventilatory behavior has used
4. Summary of Guide
freshwater fish species. This is largely because fish are gener-
ally more ecologically “visible” in their importance in aquatic
4.1 The potential toxicity of water or a pure chemical in
systems and many species (particularly the salmonids and
water is assessed by measuring changes in fish ventilatory
centrarchids) have large opercular flaps that yield relatively
behavior during exposure using a flow-through system. Sig-
clear ventilatory signals for measurement and evaluation.
nificant effects are determined by comparing specific ventila-
Species eliciting relatively small bioelectric ventilatory signals
tory responses of fish under control conditions with responses
are more difficult to use given the electrode and amplification
of those same fish during exposure conditions.Aset of control
systems referenced in this guide.
fish may also be used in the test design in order to evaluate
non-toxic changes in ventilatory response over time, particu-
5.5 Changes in ventilatory behavior have been shown to be
larly when longer-term monitoring is desired.
a reliable indicator of accidental toxic spills or “slugs” of
pollutants in wastewater and drinking water systems (15, 20,
4.2 Ventilatory responses are observed by using non-
23, 24, 33).
invasive metallic or chemically-based electrodes, a signal
amplification and filtration system, and strip chart recorder (or
6. Safety Precautions
other recording device) to display the ventilatory waveform. In
short-term tests (<24 h in duration), changes in ventilatory 6.1 Many substances may pose health risks to humans if
behaviortoexposureofatestmaterialcanbeanalyzedafterthe adequate precautions are not taken. Information on toxicity to
test by manually analyzing strip chart recordings of the humans, recommended handling procedures, and chemical and
waveform elicited over time by each fish. In experiments >24 physicalpropertiesofthetestmaterialshouldbestudiedandall
h in length or in continuous real-time monitoring applications, personnel informed before an exposure is initiated.
ventilatory waveform data are aquisitioned, analyzed, and (Warning—Specialproceduresmightbenecessarywithradio-
stored via a microcomputer equipped with an analog to digital labeled test materials and with test materials that are, or are
processor, disk or magnetic tape storage, and appropriate suspected of being carcinogenic.)
E1768 − 95 (2013)
6.2 Many materials can adversely affect humans if precau- ventilatory signal is received and recorded. Since reception of
tions are inadequate. Contact with test material, sediments, and the bioelectric signal is dependent on there being a polarity or
water should be minimized. Where appropriate, protective electrical gradient between the electrodes, electrodes are
gloves,laboratorycoats,aprons,protectiveclothing,andsafety placed opposite each other in the monitoring chamber to
glasses should be worn and dip nets, sieves, or tubes should be achieve maximum sensitivity. Several different electrode ar-
used to remove test organisms. When handling potentially rangements have been utilized including top and bottom of the
hazardous materials, proper handling procedures may include chamber (see Fig. 2(a) and (12, 15, 21)), front and back (29),
manipulating test materials under a ventilated hood or in an and sides of the chamber (see Fig. 2(b) and (11, 14, 20)). Each
enclosed glovebox, enclosing and ventilating the exposure of these arrangements may have advantages and disadvantages
chambers, and use of respirators, aprons, safety glasses and in terms of signal reception and the ability to detect subtle
gloves. changes in amplitude, body movement, or cough rates. Infor-
mation at this time suggests that a top and bottom electrode
7. Responses Measured
arrangement (see Fig. 2(a), will reduce ventilatory signal
alteration due to changes in fish position relative to the
7.1 Ventilatory parameters in fish that have been shown to
electrodes in comparison with a side electron orientation (12).
be affected by toxicity include ventilatory rate (opercular
Test chambers must be clean prior to testing as described in
movement over time), depth of ventilation (amplitude), cough-
Practice E729, and made of appropriate construction materials
ing or gill purge rate, and erratic episode frequency due to
such as glass or plexiglass (see Guide E1241 and Practice
sudden movement of the organism. Most commonly, changes
E729).
inventilatoryrate(F )havebeenusedasabioindicatoroftoxic
v
conditions (11, 12, 13, 19, 20, 21, 30, 31, 33, 34, 35, 36).
8.3 Test organisms and chambers must be isolated so as to
However, depth of ventilation and cough rate have been
reduce external stimuli such as experimenter movement,
reported to be more sensitive indicators of toxicity for some
vibration, and visual cues. This is generally achieved by
compounds (11, 19, 38, 39, 40).
placing a single fish in each chamber and by placing opaque
dividers between test chambers. The entire system (all test
7.2 Manually, changes in ventilatory rate are often deter-
chambers) should be isolated within a light-proof box or
mined by changes in the number of peaks per unit area on a
continuous light compartment.
strip-chart recording. Depth of ventilation (tidal volume) or
signal amplitude, is measured from top to the bottom of the 8.4 The electrical signal (microvolts), generated by ventila-
waveform (see Fig. 1). tory movements, that is received by the electrodes, must be
conditioned prior to use. First, the electrical components of the
7.3 Cough rate has been more difficult to determine because
system must be properly grounded to avoid erratic signal
several different types of coughs may be evident, each with its
reception. Second, electrical noise, particularly that arising
own characteristic wave form pattern (see Fig. 1 and (11, 39,
from normal 60 cycle electrical current (such as from lights,
40)). Also, without the use of video techniques, (11, 41), the
strip chart recorder, and amplifier), must be eliminated so that
actual occurrence of a cough is not always clear. Researchers
the fish ventilatory signal is received with minimal interfer-
who have investigated cough responses have interpreted most
ence. Third, the bioelectric signal must be amplified from
abnormalpeaksorpatternchangesonastrip-chartrecordingas
microvolts to millivolts in order to electrically record the
a cough. Work by Diamond et al. (11) however, indicated that
signal.Acapacitorisalsousuallyrequiredtoreduceventilatory
many of these changes may in fact be due to general activity,
signal offset from the baseline (
...