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Long-term toxicity to aquatic invertebrates

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Two long-term daphnia studies are available. One for 2,2'-(C16-18 (even numbered) alkyl imino) diethanol (CAS no.: 1218787-30-4; EC no.: 620-539-0) and one for (Hydrogenated tallow) bis(2-hydroxyethyl)amine (CAS no.: 90367-28-5; EC. no.: 291-276-3). Both substances are one and the same substance. The only difference is that the naming and CAS/EC number was updated.


Both studies are performed semi-statically 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 UVCB 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 two available long-term tests were as indicated performed following two different approaches. According to the WAF approach and

According to the bulk approach.


The WAF approach is more focused on determining the intrinsic toxicity (for C&L purposes) where the Bulk approach is more suited to derive a realistic risk ratio: PECaquatic bulk/PNECaquatic bulk as described in ECETOC Technical Report “Environmental Risk Assessment of difficult substances” (TR 88, 2003). 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. More details on the bulk approach are included in IUCLID chapter 13.


The 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 0.100 – 0.316 – 1.00 – 3.16 – 10.0 mg/L (separation factor 3.16), corresponding to the time weighted mean measured test item concentrations 2.10 – 5.30 – 8.50 – 46.0 – 87.6 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. 

The test results are presented based on nominal test loadings and on time weighted average (TWA) measured concentrations. The analytical verifications of selected components is as indicated considered 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. The test item concentrations of PFAEO-HT were analytically verified via LC-MS/MS four 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. Additional, samples with test media, but without daphnids and algae, were prepared for the sampling of the old media at the end of an exposure-renewal interval (day 1).

The C16 and C18 constituents representing 96% of the test item were used for quantification. The EL10/EC10 for reproduction after 21 days is 84/1.2 µg/L. The EL50/EC50 for adult mortality after 21 days is 1577/19.7 µg/L.
The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met.



In a further set up of the WAFs, the behavior of the test item was investigated in terms of the total fraction and the truly dissolved fraction, which was produced by a centrifugation step. The test item concentrations of PFAEO-HT were analytically verified via LC-MS/MS in the fresh media at the start of an exposure renewal interval (0 hours) as well as in the old media at the end of the exposure (24 hours) in all WAFs.
The findings were:1) In the lower nominal loading rates, the results of the truly dissolved fraction partly fell below the limit of quantification. 2) A mass balance, calculated as the sum of the results of the truly dissolved fraction and the adsorbed fraction from the centrifuge tubes, shows that the desorption of the analytes from the centrifuge tubes is not complete. A large fraction of the test item is lost during the centrifugation step.

Conclusion: The quantification of the truly dissolved fraction is not possible for 2,2’-(C16-18(even

numbered) alkyl imino) diethanol (CAS: 1218787-30-4) at the test concentrations applied.

Finally, a fingerprint was performed with the highest loading rate (10 mg/L), control and these were compared with an analytical standard of the test item prepared in acetonitrile and diluted to 10 mg/L with dilution medium. Both were verified via MS and evaluated by the software. The solutions were analytical verified via high resolution MS and evaluated by the software. In general, the concentrations and solubility of the test item is too low for significant MS spectra.


The test solutions for the Bulk approach were prepared by diluting a stable dispersion 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, 32,100, 320 and 1000 µg/L (factor √10). The test item was analytically verified in the concentration

levels 32.0 and 320 μg/L and in the control via UPLC-MS/MS. 0, 10, 17 (fresh media) and on days 3 (old media, 72 hours),

12 and 19 (old media, 48 hours).

The recoveries in the fresh media were in the range of 98 to 140 % of the nominal values. In the

old media (after 48 h or 72 h) the recoveries decreased to values in the range of 32 to 83 %.

Biodegradation as possible reason for this is very unlikely considering the short time frame

between the refreshments of the test solutions. No adsorption (< LOQ; day 10) of the test item to

the glassware was observed. The adsorbed fraction of the test item concentration was

determined to be < 9 % of the nominal concentration at the concentration level 32.0 μg/L. The

limited concentration decrease between fresh and old media is therefore most likely caused by

thermodynamically more favourable redistribution of the sorbed fraction resulting in an additional

sorption to suspended matter and DOC. The results of the chemical analyses show that the test

organisms were fully exposed to the test substance during the test. Therefore, all effect values

given are based on the nominal test item concentrations.Based on the study result, the NOEC for reproduction, mortality, adult length and weight was determined to be 320, 320 and 1000 µg/L respectively. No EC50 for parental mortality could be derived.



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 (NOECbulk/NOELWAF) to daphnia is 320/84 is 4.


  • Bulk approach: See chapter 13 IUCLID.

  • 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:
Result is based on nominal loading rates according to WAF approach. At the extreme low concentrations applied it is not possible to quantify the truly dissolved concentrations reliably. A centrifugation step of standards showed a mean loss of a factor 10.
Effect concentration:
84 µg/L

Additional information