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Long-term toxicity to aquatic invertebrates

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Description of key information

There is one long-term daphnia study available for 2,2'-(octadecylimino)bisethanol (PFAEO-C18, New CAS no.: 10213-78-2).


The study is performed According to the WAF approach, semi-statically with daily renewal of the test solutions according to OECD TG 211 with specific chemical analysis (LC-MS/MS) under GLP conditions.


Primary fatty amine ethoxylates (PFAEO) are cationic surfactants. Aquatic ecotoxicity testing of cationic surfactants is complicated as these substances are in most cases multicomponent mixtures (UVCB’s) with a range of relatively low water solubilities which sorb to equipment and organisms. 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.

OECD Guidance Document 23 on aqueous-phase toxicity testing of difficult test chemicals (Feb. 2019), advises to use the Water Accommodated Fraction (WAF) approach for these difficult substances. The new aquatic ecotoxicity studies were therefore performed according to this approach.


Per definition of the WAF approach, 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 were 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 PFAEO were as indicated in OECD GD 23 analytically verified via LC-MS/MS during the tests in the fresh media at the start of an exposure-renewal interval as well as in the old media at the end of an exposure-renewal interval. Quantification of the dissolved concentration of the test substance is however problematic for cationic surfactants especially in test solutions where algae are present. Cationic surfactants were observed to sorb strongly to algae (van Wijk et al., 2009). Removal of algae from the test solutions requires either filtration or centrifugation and at the very low test concentrations applied there is a relatively large loss (upto ±90%) of substance due to the required separation step, despite of precautions taken to limit the loss e.g. by the rinsing of the equipment.  This means that an accurate quantification of the dissolved concentration in these studies is not possible. Ecotoxicity testing with these difficult substances according to the WAF approach leads for algae and long-term daphnia tests to test results which are poorly reproducible and are associated with high uncertainty. In addition, daphnids are in the long-term test due to the strong sorption to food algae mainly exposed to PFAEO via ingestion of algae (secondary exposure route) which means that focusing only on the dissolved concentration for the dose response will lead to unrealistically low EC values for these studies.


The Daphnia magna reproduction test (semi-static, 21 d) was performed with 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2) according to OECD 211 (2012). Ten daphnids, held individually, were used per WAF loading rate and control. At the start of the exposure the daphnids were less than 24 hours old. The study was carried out under semi-static conditions with a daily renewal of the test solutions. Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, the number of broods with living juveniles, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.

The test item is a difficult substance which is strongly sorbing and poorly soluble in water. The aqueous media were prepared following OECD guidance document No. 23 (2019), which is specifically developed for aquatic toxicity testing of difficult substances, by mixing the test item with water 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 a siphon. This procedure was followed for each renewal of the test solutions. Five WAFs were prepared and tested at nominal loading rates 1.56 – 3.13 – 6.25 – 12.5 – 25.0 mg/L (separation factor 2), corresponding to the time weighted mean measured test item concentrations 1.53 – 1.63 – 2.05 – 3.30 – 6.34 µg/L.

The WAFs were checked for any undissolved or emulsified material by Tyndall effect, which was negative. The test containers were pre-treated with the appropriate test solution for at least 12 hours under test conditions.

The concentration of the test item PFAEO-C18 was quantified by measurement of the C18 Amine + 2EO constituent. The test concentrations were analytically verified via LC-MS/MS three 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 7 and 14 without daphnids and algae) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 8 and 15 with daphnia and algae) in all WAFs and in the control. Additional, samples with test media, but without daphnids and algae, were prepared for the sampling of the old media at least one time during the test (24 hours).

The stability of the test item under test conditions was monitored after 24 hours by quantifying the concentration of a parallel sample (second highest test concentration) which was prepared without daphnids and algae.

The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met.

The C18 constituent which was used for quantification represents 95.38% of the test item. The EL10/EC10 for reproduction after 21 days is <1570/1.53 µg/L. The EL50/EC50 for adult mortality after 21 days is 5230/2.24 µg/L. The NOEL/NOEC for reproduction after 21 days is <1570/1.53 µg/L. The study will therefore be repeated or the test concentration range extended. The reason for this repetition lies as indicated before in the poor reproducibility.


Additional analytical work was performed to investigate the impact of the centrifugation step on concentration of the test solutions. This was done by quantifying the WAF test solutions (without algae) before and after centrifugation. The WAFs used for this investigation were checked for any undissolved or emulsified material by Tyndall effect, which was negative.  The results of this additional work is presented in the table below.


Sampling date

Day 0


WAF test solutions 

Nominal loading

Before centrifugation

After centrifugation

Rate of test item










































limit of quantification (0.00150 mg/L of the test item)


lowest calibration level (0.0005 mg/L)



The results of the additional analytical work show that at the low test concentrations applied, the loss of substance due to the centrifugation step leads to concentrations in the supernatant which are below the Limit of quantification. For the highest nominal concentration this is a loss of >80%. It is therefore concluded that at these low test concentrations it is not possible to reliably quantify the truly dissolved concentration of 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2) in a long-term daphnia test.


Finally a finger print was performed. The peak distribution of the WAF was analyzed in fresh prepared medium in the highest test item concentration 25.0 mg/L and the control. An analytical standard of the test item was prepared in methanol and diluted to 100 µg/L with dilution medium. The highest test item concentration and the control were diluted a factor 2 with acetonitrile containing 0.2% formic acid to avoid an inhomogeneous sample. The standard dilution and the test item dilution were analytical verified via MS and evaluated by the software. The detected signals of the analytical standard and of the test item solution were compared. The concentration of the analyte C18 Amine + 2EO (358.45 Da +- 0.5 Da) in the highest test item concentration are too low for significant signals. The mass was found in the spectrum of the standard. The detected signals of the analytical standard and of the test item solution were compared and are quite similar. In the analytical standard the mass 358.45 m/z is shown and related to the test item. In general, the concentrations and solubility of the WAF are too low for significant MS spectra.



  • 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:
Test performed according to the WAF approach. EL10 based on loading rate.
Effect concentration:
< 1 570 µg/L

Additional information

The 21d EC50 for adult mortality of 566 µg/L may be used as a short term endpoint as a worst-case.