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EC number: 931-292-6
CAS number: 308062-28-4
This discussion describes the
rationale for the conduct of higher tier, periphyton microcosm studies
and how these support an Application Factor (AF) between 1-10 applied to
the NOEC result depending
on the assessment situation. The
information is supplemental to the publications of Belanger et al.
(1996), Belanger et al. (1997) and ECETOC (1997). In
the case where potential algal toxicity indicated by single species
inhibition assays for a particular chemical drives the environmental
risk assessment, an algal periphyton bioassay may have increased
relevance, and if conducted well AFs between 2-5 may be more appropriate.
According to experts in the
field of ecotoxicology and model ecosystem (microcosm and mesocosm)
science, the derivation of PNECs (Predicted No Effect Concentrations) is
based on the development of a study to be “fit for purpose”. Defining
the purpose is difficult and requires expert judgment and the
application of sound ecotoxicological science. These
are articulated in several documents summarizing expert consultations
and meetings in which a wide variety of microcosm and mesocosm (also
known as semi-field tests and model ecosystem tests) were discussed and
presented (Crossland et al. 1992; ECETOC 1997; Giddings et al. 2002,
OECD 2006). ECETOC
(1997) articulated the most important aspects of model ecosystems that
contribute to assignment of no or low application factors. Each
factor exists on its own scale and each study or model ecosystem is
judged by its own merits, therefore supporting the statements in theTGD
(ECHA 2008) indicating assignment of AFs occur on a case-by-case basis. From
this point of view, flowing water model ecosystems should be considered
distinct from pond or standing water mesocosms commonly used to evaluate
fate and effects of pesticides. In
accordance with the expected environmental exposure of high production
volume chemicals with wide, dispersive use and discharge to rivers or
seas, flowing water model ecosystems are inherently better fit for this
purpose than standing water (lentic) model ecosystems for these types of
Table 1. Summary
of periphyton community bioassay conditions (see Belanger et al. 1996
0.5 m long, 2-3 L volume
Source of biota
field colonized, minimum of 6-8 weeks
Source of additional colonists
Filamentous, expression of slower current communities over time
Growlux, 15 hr on/9 hr off @ 530 ± 60 µE/m2/sec
blended well water delivered by Mount-Brungs proportional dilutor, > 15 turnovers per day
Duration of study
28 d dosing after colonization
Surface area of individual colonization substrate
Velocity of water
Slow (1 volume replaced/1.5 hr)
usually 3 per concentration; 5 exposures with control
Population and community structure; only autotrophs
Sensitivity of biota
Most sensitive taxa based on single species testing
Specific methods, performed weekly in all chambers
The factors ECETOC (1997)
considered suitable for model ecosystem assessments include biological
complexity, sensitivity, study duration, exposure determination, and
relevance to natural systems. The
typical system and experimental design are summarized in Table 1.
Below is a summary of the
status of the periphyton microcosm bioassay with respect to the primary
factors identified by ECETOC (1997).
Complexity – the periphyton microcosm contained a highly diverse and
complex attached algal community
algal species were normally present
system is dominated numerically by diatoms
green algae are common upon which additional epiphytic (algae attached
to other algae and not the inert substrate) taxa reside
10-15 would be numerically dominant, typical of natural algal communities
spread across 6 – 8 major phyla (most commonly Bacillariophyta
[diatoms], green algae, blue-green algae, Euglenophgyta, Rhodophyta [red
algae], and Chrysophyta)
included those that were obligate epiphytes (must exist on other algae),
periphyton (generally attached to anything submerged), lithophytes
(attached to rock), freely mobile, stalked, and filamentous species. The
community was fully three-dimensional.
some assays, multiple communities derived from separate colonization
sites were tested
simultaneously to understand the relative importance of geographic and
water quality driven influences on test outcomes. In
these cases, breadth of the tested biota was greater.
– the periphyton microcosm was optimized for statistical and biological
endpoints evaluated possessed Minimum Detectable Differences using
inferential statistics of 10-25% (change needed to be identified as
statistically different from the control) including community diversity
and population abundance measures.
algae were diatoms, many were known sensitive species, based on single
species toxicity literature and other microcosm and mesocosm
investigations (e.g., the diatom Melosira varians).
endpoints were also investigated including community metabolism
(respiration plus photosynthesis) and degradation. However,
these are known to be less sensitive than structural measures of effects.
– the periphyton community bioassay was approximately 2 months duration,
longer than algal chronic toxicity tests
of the tile substrates was for 5 weeks; colonization of natural
substrates was undoubtedly longer but not controlled.
were conducted over long durations (28 days) relative to the life span
of individual algae or the typical algal toxicity test. Multiple
assessment time points were used to judge the ecological trajectory and
stability of the populations and communities.
– the exposure to test substances was verified at least weekly in
treatment units and stock solutions
analytical confirmed a high % of nominal. The
test system includes microbial degraders (algae, fungi, and bacteria)
which result in deviations from nominal for biodegradable substances.
system was designed to be flow through, typically exceeding 15 turnover
volumes in each exposure chamber per day. This
is at the limit of what most proportional dilutor systems can deliver.
to Natural Systems – the periphyton communities were nearly
indistinguishable from natural systems and was representative streams
that were relatively uninfluenced by man
situ colonization in a high quality receiving water and provided the
algae for testing. Substrates
were colonized in the upper Little Miami River, a National Wild and
Scenic river of the(see http://www.dnr.state.oh.us/dnap/sr/lmiami/tabid/1862/Default.aspx). Similarly,
this is also a National Wild and Scenic river (see http://www.dnr.state.oh.us/tabid/981/default.aspx). Therefore,
the algal periphyton colonized for testing in the bioassay come from a
minimally impacted system known to contain sensitive species. Results
should be protective of a wide variety of river systems (impacted and
rivers contain a number of threatened and endangered species (fish and
mollusks) (over a dozen in each) .
Little Miami river reach used in this study has been studied for several
decades (see also Lewis 1986; Lewis et al. 1986a; Lewis et al. 1993;
Belanger et al. 1996) and its algal assemblages are thus well described. The
system has been well studied as well, particularly in an ecological risk
assessment context (Schubauer-Berigan et al. 2000).
the algal communities used in the periphyton community bioassay are
representative of temperate river communities (Lowe 1974) and are
typical of a globally distributed, cosmopolitan microbial group (Cairns
et al. 1986). The
type localities for many of the species described in the study are
European (,,, etc.).
The assignment of a low
application factor to a particular study is a combination of evaluation
of the attributes of the test system, assessment of important endpoints
for a particular study and compound, relevance of the study to the
discharge situation, and best professional judgment. From
the perspective of a microcosm, additional care needs to be given to the
assignment of an application factor and should be done in concert with
known data from single species toxicity tests. For
example, if the invertebrate Daphnia magna was the most sensitive
in single species tests, a microcosm that did not contain perspective on
invertebrate sensitivity would be uninformative and the result would not
provide additional information in the risk assessment. However,
if algae were the most sensitive taxonomic group in single species
tests, and the microcosm provided detailed additional information on the
tolerance and sensitivity of algae to the chemical of interest, this can
be used to further inform the environmental risk assessment.
The Case for a Periphyton
Community Bioassay on Amine Oxide (AO)
The following points should
full study, from proposal through final reporting, was performed under
an independent Quality Assurance Unit. Protocol,
in-life audits, and study report audits were completed and reports to
P&G management are noted in the full study report. The
study records are permanently archived in P&G.
tested AO was a commercially relevant form (a mixture of C12, 14, and 16
chain lengths at 70, 25, and 5% weight contribution, respectively,
described in the report) ;
AO was dosed in a laboratory well water at maximum free dissolved
concentration, below the solubility limit;
periphyton bioassay on amine oxide evaluated 3 different periphyton
communities (essentially three different tests) assessed simultaneously. Details
are found in the study report.
different source river systems (Little Miami River,)
colonized substrates (natural cobble, terra cotta clay tile)
communities were biologically very complex and allowed quantitative
probing of the sensitivity of 18 different species at the population
richness (number of different species observed per individual sample) at
each time point averaged between 35-45 species.
tested communities were ecologically mature and representative of
natural communities. Dominant
species were consistently recovered over the duration of the experiment. This
strongly suggests that the communities were self-perpetuating and not
adversely influenced by being dosed in well water and housed in the
indoor test system.
studies involved exposure of AO for 28 days, 7 times longer than a
typical algal chronic toxicity test. Communities
were mature at the time of testing which is a function of the duration
of colonization (35 days), time of year, and location.
included population and community level structural endpoints, known to
be more sensitive than functional endpoints (community rate processes
such as respiration and photosynthesis) (Giddings et al. 2002).
The NOEC for Amine Oxide for
all three tested communities was 67 µg/L. Each
community had unique constituent species; however, several were
that single species algal inhibition tests clearly identify algae as the
most sensitive trophic level, the complexity of the periphyton community
microcosm bioassay is very high, is of very long duration, and the
system is sensitive, an application factor of 2 appears justifiable.
Algae are the most sensitive species for
aquatic toxicity. The classification is derived using the ErC50 of 0.143
mg AO/L and the NOEC of 0.015 mg AO/L, which are geometric mean values
calculated from the results of the four studies available for
Pseudokirchneriella subcapitata. On the basis of the ErC50 the substance
is classified for acute/short-term aquatic toxicity as Acute Category 1.
As the ErC50 is between 0.1 and 1, a Multiplying Factor (M) = 1 applies.
On the basis of the NOEC and taking into account the ready
biodegradability of amine oxides, the substance is classified for
long-term aquatic toxicity as Chronic Category 2.
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