2,2'-(octadecylimino)bisethanol

Toxicity to aquatic algae and cyanobacteria

Description of key information

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

 

Primary fatty amine ethoxylates (PFAEO) are cationic surfactants for which aquatic ecotoxicity testing 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 for the PFAEO 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.

 

As indicated the test item is a difficult substance with a low water solubility and therefore like for the other primary alkyl amine ethoxylates Water Accommodated Fractions (WAF) were prepared in agreement with OECD GD 23 (2019).

The toxicity of 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to OECD TG 201 and EC method C.3 under GLP conditions.

The study was conducted under static conditions with an initial cell density of 5375 cells/mL. Five water accommodated fractions (WAFs) were prepared 24 ± 1 hour prior to the start of exposure with nominal loading rates of the test item in the range of 0.0400 to 4.00 mg/L set up in a geometric series with a factor of √10: 0.0400 - 0.127 - 0.400 - 1.27 – 4.00 mg/L. The measured concentrations were in all loading rates < LOQ. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The test media were visually clear at the beginning of the test. The concentrations of the test item 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2) 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. Additionally, the loading rate 0.400 µg/L was analytically verified without algae determined 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. The test item is poor soluble in water and low concentrations at the start of the exposure could be observed. After 24 hours no further test item could be detected. The test item could not be detected in the algae and glassware extracts.

All effect values are given as nominal loading rates see Table 1.

 

Table 1: NOEL, LOEL and ELx-values and 95% Confidence Intervals of 2,2’ (octadecylimino) bisethanol (CAS: 10213-78-2) (0 - 72 hours) based on the nominal test item loadings [mg/L]  

	Inhibition of Growth Rate
NOEL	0.127
LOEL	0.400
ErL10	0.772 (0.387 – 1.04)
ErL20	1.26 (1.07 – 1.51)
ErL50	2.56 (2.09 – 3.26)
	Inhibition of Yield
NOEL	0.0400
LOEL	0.127
EyL10	0.0355 (< 0.0400 – 0.0563)
EyL20	0.0771 (0.0460 – 0.131)
EyL50	0.607 (0.444 – 0.852)

 

The exposure concentrations were quantified by analyzing the main constituent 2,2” (octadecylimino) bisethanol which represents 95.38% of the total active ingredient.

 

 

 

Additional analytical work was performed to investigate the impact of the centrifugation step on the WAF test solutions. This was done by quantifying the WAF test solutions (without algae) before and after centrifugation. 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
[mg/L]	Meas.	%	Meas.	%
	conc.		conc.
	[mg/L]		[mg/L]
8	0.00196	0	< LOD
2.53	0.00135^1)	0	< LOD
0.8	0.00120^1)	0	< LOD
0.253	0.00105^1)	0	< LOD
0.08	< LOD		< LOD
Control	< LOD		< LOD
LOQ	limit of quantification (0.00150 mg/L of the test item)
LOD	lowest calibration level (0.0005 mg/L)
1)	value is < LOQ but above LOD

 

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 detection. For the highest nominal concentration this is a loss of >74%. 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 an algae test.

 

The calculated ErL10 and ErL50 values with 95 % confidence intervals for inhibition of specific growth rate were 772 (387 – 1040 )and 2560 (2090 – 3260) μg/L, respectively.

 

 

• 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

EC50 for freshwater algae:

2.56 mg/L

EC10 or NOEC for freshwater algae:

0.772 mg/L

Additional information

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), advices to use the Water Accommodated Fraction (WAF) approach for these UVCB substances. The 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 (frequency depending on test type). 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 requires either filtration or centrifugation and at the very low test concentrations applied there is a relatively large loss (factor ±10 concentration reduction) 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 which means that focusing only on the dissolved concentration for the dose response will lead to unrealistically low EC values for these studies.