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Long-term toxicity to aquatic invertebrates

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Description of key information

Two long-term daphnia studies are available for:

New Name: 2,2'-(octadec-9-enylimino)bisethanol (CAS no 25307-17-9)

Old Name: Bis(2-hydroxyethyl) oleyl amine

Abbreviation: PFAEO-O


Both studies are performed to evaluate the long-term toxicity to aquatic invertebrates but follow two different approaches.


PFAEO-O is a multicomponent mixture (UVCB) of cationic surface-active constituents with different water solubilities. The fate of cationic surfactants in general deviates from standard chemicals. These substances are therefore considered as difficult substances for which the results of standard guideline studies are very difficult to interpret when considering them in a standard way. The reasons are the intrinsic properties like the relatively low water solubility and strong sorption to equipment and organisms. Classical ecotoxicity testing with these substances using reconstituted water often leads to test results which are poorly reproducible and are associated with high uncertainty. In addition, because of the complex sorption mechanisms (van der Waals and Ionic mechanisms) the actual dissolved exposure concentration cannot reliably be estimated.

The two available long-term tests where therefore performed following two different approaches.


One test which is focused on determining the intrinsic toxicity of PFAEO-O (for C&L purposes) is performed according to the Water Accommodate Fraction (WAF) approach as described in “OECD guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (No. 23 Feb. 2019) with a daily refreshment of the test solutions. The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration.


The other test which is more suited to derive a realistic risk ratio for the aquatic compartment is performed according to the PECaquatic bulk/PNECaquatic bulk approach as described in ECETOC Technical Report “Environmental Risk Assessment of difficult substances” (TR 88, 2003) with a three times a week refreshment of the test solutions. The so called “Bulk approach” is used in the environmental risk assessment to cope with the earlier mentioned lack of realistic PEC estimation. Instead of using the dissolved PECwater, the Bulk concentration (dissolved + sorbed) in water is used. This bulk approach requires a PNECwater, bulk that means that testing has to use river water which contains dissolved organic carbon and suspended organic and inorganic matter, instead of reconstituted water.

Tests according to the bulk approach were thus performed because the partitioning of cationic surfactants to soil, sediment or suspended matter is rather complex which explains why there is no alternative Equilibrium Partitioning Method (EPM, di Toro, 2008) formula for these substances available yet. The use of the Bulk approach however elegantly bypasses this deficiency as it eliminates the EPM on the exposure and effect side.


Main difference between the two approaches lies in the preparation of the test solutions and how the results should be interpreted.

  • For the preparation of the test solutions according to the WAF approach, all reasonable efforts were taken to produce a solution of all soluble components of the test item in test media. The test solutions were prepared daily, by gentle mixing the test item with test medium for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphoning. This procedure was followed for each renewal of the test solutions. Five WAFs were prepared and tested at nominal loading rates 00 – 10.0 – 25.0 – 62.5 - 156 µg//L (separation factor 2.5), corresponding to the time weighted mean measured test item concentrations 0.850 – 1.79 – 5.45 – 24.5 – 66.9 µg/L.
    No undissolved or emulsified material was observed in the WAF solutions based on the Tyndall effect check. Adsorptive losses to the glass test vessels were kept as low as possible by pre-conditioning the test vessels already with appropriate test solution for at least 12 hours under test conditions. Before the start of the exposure and each renewal, the test containers were emptied and refilled with freshly prepared test solutions. 

  • It should be noted that the test substance sorbs strongly to the food algae (van Wijk, 2009) which leads to an apparent reduction of the freely dissolve concentration (when algae are separated prior to analysis) but are still providing a secondary exposure route via ingestion. 

  • The results are presented based on nominal test loadings and on time weighted average (TWA) measured concentrations. The EL10/EC10 for reproduction after 21 days is 50.7/17.2 µg/L. The EL50/EC50 for adult mortality after 21 days is >156/>66.94 µg/L. The TWA results are given despite the fact that per definition of the WAF, all terms related to concentration level should be given as loading rates (mass-to-volume ratio of the substance to the medium) because partly dissolved compounds and mixtures cannot be related to concentrations. Analytical verifications of selected components can be helpful and deliver supporting information, but they do not represent the whole test substance and therefore, toxicity results will be evaluated based on WAF loading rate (Wheeler, Lyon et al. 2020). Several guidance documents suggest to use the WAF loading rate for the environmental hazard classification of chemical substances e.g. the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (OECD 2002, OECD 2019) as well as OECD guidance documents on the classification of chemicals which are Hazardous for the Aquatic Environment. The test item concentrations of 2,2’-(Octadec-9-enylimino)bisethanol were analytically verified via LC-MS/MS 3 times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7 and 14) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8 and 15) in all WAFs and in the control.
    The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met.

    A fingerprint was performed with the highest loading rate (156 µg/L and compared with the analytical standard with the same concentration of the test item prepared in methanol. Both were verified via MS and evaluated by the software. The solutions were analytical verified via high resolution MS and evaluated by the software. The detected signals of the analytical standard and of the test item solution were compared. In test item and standard solutions the mass of C18:1 Amine + 2 EO of the test item was found (356.31 Da ± 0.5 Da). For all other components of the UVCB the content was too low for analysis.


  • The test solutions for the Bulk approach were prepared by diluting a stable emulsion of 10 mg/L in test medium. In agreement with the bulk approach the test medium used was natural surface water. The following concentrations were prepared by diluting the stock solution in test medium: 10, 30, 90, 270, and 810 µg/L. Due to the use of non-standard test medium (natural river water) the results of bulk approach test are considered inadequate by regulators involved in C&L because they do not fulfill to the narrow criteria set to quantify the intrinsic toxicity. There is however a clear difference in the evaluation of a standard aquatic ecotoxicity test and an ecotoxicity test performed using the Bulk approach. In order to class a standard laboratory toxicity study valid, it is of particular importance that – besides information on test substance, test method/conditions and test organism used - suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations are based upon measured levels of the dissolved concentration.

For ecotoxicity tests performed using the bulk approach, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware which was <LOQ, should be accounted for. For a valid bulk approach test the dose-response relationship should thus be based on the sum of adsorbed and dissolved substance. Results from bulk-approach tests are therefore easier to interpret because nominal concentrations corrected for sorption to glassware can be used to quantify the dose. Because of the use of natural river water a bulk approach test is more environmentally realistic than the standard method and due to that considered to be a higher tier study.


Droge & Goss (2013) have shown that sorption of cationic surfactants to soil and sediment is mainly driven by electrostatic interaction and to a lesser extent by hydrophobic interaction. This means that both the suspended matter and dissolved organic carbon in surface water are the key surface water properties determining the bioavailability of the test item.

The natural surface water was therefore characterized in detail and selected to contain a realistic worst-case suspended matter concentration of 15±3.5 mg/L and ± 3.5 mg/L DOC(≈NPOC). It should be noted that this composition is in perfect alignment with the risk assessment method developed by ECHA, as the concentration of suspended matter in surface water is considered to be 15 mg/L in CHESAR III for risk assessment (see ECHA’s guidance R.16, v3.0, Feb 2016, p. 88).


Sorption to glassware was observed to be <LOQ which means that the nominal test concentrations can be used for the dose-reponse because the recoveries in the fresh media were in the range of ≥80 % of the nominal values. The difference between the observed recoveries and nominal concentrations is explained by the rapid sorption of PFAEO-O to the river water constituents. Based on the study result, the EC10 for reproduction was determined to be 10.7 µg/L. The EC50 for parental mortality was determined to be 301 µg/L,


The degree of mitigation of the long-term toxicity to daphnia due to the use of natural river water can be calculated by taking the ratio of the results observed for the bulk approach test and the nominal test results observed for the WAF approach.

The mitigation factor for the chronic effect (EC10-bulk/EC10-WAF) to daphnia is 10.7/50.7 is 0.21


  • Droge, S.T.J. and Goss, K.W. (2013) Development and Evaluation of a New Sorption Model for Organic Cations in Soil: Contributions from Organic Matter and Clay Minerals. Environmental Science and Technology, 47:14233-14241.

  • Di Toro, D (2008) Bioavailability of chemicals in Sediments and soils: toxicological and chemical interactions. SERDP/ESTCP Bioavailability workshop

  • van Wijk, D., Gyimesi-van den Bos, M., Garttener-Arends, I., Geurts, M., Kamstra, J., Thomas, P., (2009) Bioavailability and detoxification of cationics, I. Algal toxicity of trimethylammonium salts in the presence of suspended matter and humic acid. Chemosphere 75 (3), 303–309.

  • OECD (2002). Guidance Document on the Use of the Harmonised System for the Classification of Chemicals which are Hazardous for the Aquatic Environment.

  • Wheeler, J. R., D. Lyon, C. Di Paolo, A. Grosso and M. Crane (2020). "Challenges in the regulatory use of water-accommodated fractions for assessing complex substances." Environmental Sciences Europe 32(1): 1-10.

  • OECD (2019): Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals. OECD series on testing and assessment no. 23 (second edition), ENV/JM/MONO(2000)6/REV1






Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
Effect concentration:
50.7 µg/L

Additional information