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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Link to relevant study record(s)

Description of key information

One toxicity test with algae was performed. The test is well described and performed according OECD guideline 201 applying GLP conditions. The test is performed in natural river water i.e. applying the bulk approach and resulted in a 72 hr ErC10 of 34.5 µg/L  and 72 hr ErC50 of 119 µg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
119 µg/L
EC10 or NOEC for freshwater algae:
34.5 µg/L

Additional information

Two algae studies are available for 2,2'-(C16-18 (even numbered) alkyl imino) diethanol (PFAEO-HT, New CAS no.: 1218787-30-4; old name: Bis (2-hydroxyethyl) hydrogenated tallowalkylamine, Old CAS No.: 90367-28-5).

  • one study is performed according to the Water Accommodate Fraction (WAF) approach and

  • one under is performed according to the Bulk-approach.


PFAEO-HT 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 which have an impact on both the effect and exposure assessment.

Effect assessment: Classical ecotoxicity testing with these substances using reconstituted water often leads to test results which are poorly reproducible and are associated with high uncertainty.

Exposure assessment: Because of the complex sorption mechanisms (van der Waals and Ionic mechanisms) the actual dissolved exposure concentration cannot reliably be estimated with the equilibrium partitioning method.


The two available freshwater algae tests where therefore performed following two different approaches. Both studies are considered to be of equal quality but are performed for different purposes.

One test which is focused on determining the intrinsic toxicity of PFAEO-HT (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). 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 better 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). The so called “Bulk approach” is used in the environmental risk assessment to cope with the earlier mentioned lack of realistic PEC estimation and problems observed with the reproducibility of the specific chemical analyses.

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 lab 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. (See chapter 13 in IUCLID for more details on the Bulk approach).


Main difference between the WAF and Bulk approach lies in the preparation of the test solutions and how the results should be interpreted. 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 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. During the test, similar as in the environment, the DOC and suspended matter present will serve as passive dosing reservoirs maintaining a more constant exposure.


Study 1:  As indicated the test item is a UVCB substance with constituents of different water solubility and therefore, in agreement with OECD guidance 23, Water Accommodated Fractions (WAF) were prepared. For this algae test seven WAFs were prepared 24 ± 1 hours prior to the start of exposure with nominal loading rates of the test item in the range of 0.00800 to 8.00 μg/L set up in a geometric series with a factor of √10: 0.800 – 2.53 – 8.00 – 25.3 – 80.0 μg/L corresponding to the time weighted average concentrations of 0.200 – 0.473 – 0.897 – 2.14 – 4.48 μg/L. For preparation of the loading rates the test item was dissolved in methanol (0.16 and 1.6 g/L). The test item with

methanol was applied onto a curved glass slide and the methanol was evaporated. The glass slide with the test item was inserted in a glass flask with an appropriate amount of dilution water. A slow stirring procedure was applied for 24 ± 1 hour at room temperature. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour, the aqueous phase of the WAF was removed by siphoning (from the approximate middle of the glass flask). The WAFs were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative. The resulting water accommodated fractions (WAF) were used in the test. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions during the test were within the acceptable limits.

The test media were clear by eye during the exposure period of 72 hours. The concentrations of

the test item 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4) were

determined at the start of the exposure (0 hours), after 24 and 48 hours and at the end of the

exposure (72 hours) of all tested loading rates and the control via LC-MS/MS with algae. Samples

after 24, 48, 72 hours of exposure were centrifuged to separate the algae from the water phase.

Both were analyzed separately to determine the truly dissolved test item fraction and the fraction

adsorbed to algae. As well as the concentration lost due to the centrifugation step. Additionally,

the loading rate 25.3 μg/L was analytically verified without algae at the start of the exposure

(0 hours), after 24 and 48 hours and at the end of the exposure (72 hours) via LC-MS/MS.As the test item is a UVCB substance, all evaluations are presented based on the nominal loading rates of PFAEO-HT.

The exposure concentrations were quantified by analyzing the 2 main constituents (C16 and C18 which together represent 95.9% of the total active ingredient). In a further set up of the WAFs (as in the definitive test) 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. Findings: 1)In all nominal loading rates the results of the truly dissolved fraction partly fell below the limit of quantification. 2) The results of centrifugation with and without algae are comparable.

Conclusion: Therefore, the determination of the truly dissolved fraction is not possible with 2,2’-(C16-18(even numbered) alkyl imino) diethanol (CAS: 1218787-30-4).

The calculated ErL10 and ErL50 values with 95 % confidence intervals for inhibition of specific growth rate were 13.9 (5.82 – 22.1) and 55.8 (35.5 – 73.5) μg/L, respectively.


Study 2: A study was conducted to determine the fresh water algal growth inhibition of the test substance, PFAEO-HT (99.9% active), according to the OECD Guideline 201, in compliance with GLP. The toxicity of the test substance to an exponentially growing culture of Pseudokirchnerella subcapitata was determined over an exposure period of 72 h with nominal concentrations of 1.00 - 3.20 - 10.0 - 32.0 - 100 – 320 µg/L. The test was carried out according to the bulk approach using enriched natural surface water with a low Dissolved Organic Carbon (DOC; 3.8 mg/L) and Total Suspended Solids (TSS; ± 17.6 mg/L) content, allowing a more environmentally realistic determination of the effects of the test substance.

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 substance.

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).


When applying the bulk approach, the truly dissolved concentration is eliminated from the PEC/PNEC equation (see IUCLID chapter 13), only confirmation that the initial exposure concentrations are within 20% of the nominal concentrations is needed and sorption to glassware needs to be taken into account.


The concentrations of PFAEO-HT in the test were quantified at the concentration

levels 3.2 and 100 mg/L (prepared without algae) and the control at test start via LC-MS/MS

analysis. The measured concentrations at test start were in the range of 104 – 132 % of the nominal

values. At the end of the test Bis (2-hydroxyethyl) hydrogenated tallowalkylamine was analysed at concentration levels 3.2 and 100 mg/L (prepared without algae) and gave recoveries of <LOQ - 16 % of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, also the river water was frozen before use to minimize the microbial activity. The decrease was attributed to additional sorption to suspended matter and DOC due to thermo-dynamically driven redistribution of the sorbed fraction. No adsorption (< LOQ) of the test item to glassware was observed. Therefore, all effect values are given based on nominal

concentrations of the test item.

Bis (2-hydroxyethyl) hydrogenated tallowalkylamine (PFAEO-HT) was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h with the following effect values

(nominal concentrations): The EC10-values with 95 % confidence intervals for inhibition of specific

growth rate (ErC10) and yield (EyC10) after 72 h were 34.5 (27.6 – 42.4) and 2.15 (<1 – 6.16) μg/L,

respectively. The EC50-values with 95 % confidence intervals for inhibition of specific growth rate

(ErC50) and yield (EyC50) after 72 h were 119 (111 – 128) and 46.9 (28.2 – 82.3) μg/L, respectively.

The mitigation factor of the river water constituents in the bulk approach test can be calculated by taking the ratio between the bulk approach effect concentrations and the WAF approach effect concentrations based on loading:  ErC10 bulk/ErL10 WAF = 34.5/13.9 = 2.5  



  • 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