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Hazard for aquatic organisms


Hazard assessment conclusion:
PNEC aqua (freshwater)
PNEC value:
0.034 mg/L
Assessment factor:
Extrapolation method:
assessment factor
PNEC freshwater (intermittent releases):
0.034 mg/L

Marine water

Hazard assessment conclusion:
PNEC aqua (marine water)
PNEC value:
0.003 mg/L
Assessment factor:
Extrapolation method:
assessment factor
PNEC marine water (intermittent releases):
0.003 mg/L


Hazard assessment conclusion:
PNEC value:
4.59 mg/L
Assessment factor:
Extrapolation method:
assessment factor

Sediment (freshwater)

Hazard assessment conclusion:
PNEC sediment (freshwater)
PNEC value:
5.24 mg/kg sediment dw
Extrapolation method:
equilibrium partitioning method

Sediment (marine water)

Hazard assessment conclusion:
PNEC sediment (marine water)
PNEC value:
0.524 mg/kg sediment dw
Extrapolation method:
equilibrium partitioning method

Hazard for air


Hazard assessment conclusion:
no hazard identified

Hazard for terrestrial organisms


Hazard assessment conclusion:
PNEC soil
PNEC value:
1.02 mg/kg soil dw
Extrapolation method:
equilibrium partitioning method

Hazard for predators

Secondary poisoning

Hazard assessment conclusion:
PNEC oral
PNEC value:
11.1 mg/kg food
Assessment factor:

Additional information

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 chemicals. 

Table 1. Summary of periphyton community bioassay conditions (see Belanger et al. 1996 for details).


Periphyton Bioassay




0.5 m long, 2-3 L volume

Source of biota

field colonized, minimum of 6-8 weeks

Source of additional colonists


Community succession

Filamentous, expression of slower current communities over time


Growlux, 15 hr on/9 hr off @ 530 ± 60 µE/m2/sec

Dilution water

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

2500 mm2

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).

1.     Biological Complexity – the periphyton microcosm contained a highly diverse and complex attached algal community

a.      50-125 algal species were normally present

i.     The system is dominated numerically by diatoms

 ii.     Filamentous green algae are common upon which additional epiphytic (algae attached to other algae and not the inert substrate) taxa reside

 iii.     Generally 10-15 would be numerically dominant, typical of natural algal communities

b.      Typically spread across 6 – 8 major phyla (most commonly Bacillariophyta [diatoms], green algae, blue-green algae, Euglenophgyta, Rhodophyta [red algae], and Chrysophyta)

c.      Taxa 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.

d.      In 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.

2.     Sensitivity – the periphyton microcosm was optimized for statistical and biological sensitivity

a.      Statistical

 i.     Key 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.

b.      Biological

 i.     Dominant 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).

 ii.     Functional 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.

3.     Duration – the periphyton community bioassay was approximately 2 months duration, longer than algal chronic toxicity tests

a.      Colonization of the tile substrates was for 5 weeks; colonization of natural substrates was undoubtedly longer but not controlled.

b.      Studies 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.

4.     Exposure – the exposure to test substances was verified at least weekly in treatment units and stock solutions

a.      Detailed 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.

b.      The 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.

5.     Relevance to Natural Systems – the periphyton communities were nearly indistinguishable from natural systems and was representative streams that were relatively uninfluenced by man

a.      In 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 Similarly, this is also a National Wild and Scenic river (see 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 uninmpacted).   Both rivers contain a number of threatened and endangered species (fish and mollusks) (over a dozen in each) .

b.      The 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).

c.      Importantly, 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 be considered:

1.      The 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.

2.      The 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) ;

3.      The AO was dosed in a laboratory well water at maximum free dissolved concentration, below the solubility limit;

4.      The periphyton bioassay on amine oxide evaluated 3 different periphyton communities (essentially three different tests) assessed simultaneously. Details are found in the study report.

a.      Two different source river systems (Little Miami River,)

b.      Two colonized substrates (natural cobble, terra cotta clay tile)

5.      The communities were biologically very complex and allowed quantitative probing of the sensitivity of 18 different species at the population level. Taxa richness (number of different species observed per individual sample) at each time point averaged between 35-45 species.

6.      The 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.

7.      The 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.

8.      Endpoints 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 overlapping. Considering 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.

Conclusion on classification

From the studies available for a number of amine oxides, algae are the species most sensitive to these substances.

The Acute classification is derived using the ErC50 of 0.16 mg/L which is the geometric mean of the results from studies performed with C10 AO, C12 AO, C14 AO and C12-14 AO on Pseudokirchneriella subcapitata which appears to be the most sensitive algal species based on the available data. 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.

A NOEC is not available from the studies performed using C10 AO, C12 AO or C14 AO hence the Chronic classification is derived using the NOEC of 0.015 mg AO/L, which is the geometric mean value calculated from the results of the four studies performed with C12-14 AO on Pseudokirchneriella subcapitata. 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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