Reaction mass of 2,2',2''-nitrilotriethanol and 2,2'-[[2-[(2-hydroxyethyl)amino]ethyl]imino]bisethanol and 2,2'-iminodiethanol

Toxicity to aquatic algae and cyanobacteria

Description of key information

The main constituent triethanolamine shows low toxicity to algae. The constituent DEA, present at 25% is the most toxic component From the data availabel from Dithanolamine the following vvalues are calculated:

EC50 freshwater algae 9.5/0.25 = 38 mg/L

Marine algae EC50 86.96/0.25 = 348 mg/L

EC10 freshwater algae = 1.1/0.25 = 4.4 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

38 mg/L

EC50 for marine water algae:

348 mg/L

EC10 or NOEC for freshwater algae:

4.4 mg/L

Additional information

Regarding the toxicity of DEA to algae, data of different studies and screening tests are available covering the freshwater species Ankistrodesmus bibraianus, Chlorella vulgaris, Desmodesmus subspicatus, Microcystis aeruginosa, Pseudokirchneriella subcapitata and Scenedesmus quadricauda as well as the marine species Phaeodactylum tricornutum and Skeletonema costatum. The respective results are summarized in the table below and subsequently discussed in detail:

Table: Effect Concentrations of DEA, determined for aquatic algae:

	Guideline	Species	Results [mg/L]
Key Study Dow Chemical (1982)	US EPA algal assay bottle test 600/9-78-0718	P. subcapitata	Recalculation with ToxRat v2.10: Test 1: 72-h NOErC: 0.6 72-h ErC10: 1.4 72-h ErC50: 9.5 Test 2: 72-h NOErC: n.d.* 72-h ErC10: 1.1. 72-h ErC50: 10 Original result: 96-h ErC50: 2.2 (geom. mean)
Supporting Study NAPM (1974)	no guideline followed (US EPA provisional algal assay procedure, PAAP)	P. subcapitata	7-d NOErC: 10 7-d ErC50: > 10 - < 100
Supporting Study Zurita et al. (2005)	no guideline followed; cell multiplication inhibition test;	C. vulgaris	72-h ErC50: 778
Supporting Study Bringmann & Kuehn (1978 a, b)	no guideline followed; growth inhibition test according to Bringmann	M. aeruginosa	Not neutralised: 8-d (TTC)** ErC3: 17 Neutralised: 8-d (TTC)** ErC3: 16
Supporting Study Bringmann & Kuehn (1977 - 1980)	no guideline followed; growth inhibition test according to Bringmann	S. quadricauda	Not neutralised: 8-d (TTC)** ErC3: 10 Neutralised: 8-d (TTC)** ErC3: 4.4
Screening Tests (supporting study) BASF AG (1992)	DIN 38412-9; Re-evaluated with ToxRat v2.10	D. subspicatus	Not neutralised: 72-h ErC10: 85.7 72-h ErC50: 107.3 Neutralised: 72-h ErC10: 2.4 72-h ErC50: 14.9
Screening Tests (supporting study) BASF AG (1992)	DIN 38412-9; Re-evaluated with ToxRat v2.10	A. bibraianus	Not neutralised: 72-h ErC10: 30.1 72- ErC50: 57.5 Neutralised: 72-h ErC10: 123.9 72-h ErC50: > 100***
Marine test (supporting study) Libralato et al. (2010)	ISO 10253; Marine algal growth inhibition test	P. tricornutum(marine)	72-h NOErC: < 16 72-h ErC50: 86.96
Marine test (supporting study) Cowgill et al. (1988)	no guideline followed (US EPA marine algal assay procedure, bottle test, 1974)	S. costatum(marine)	5-d NOErC: 216 5-d ErC50: 548.2
Marine test (supporting study) Eide-Haugmo et al. (2012/2009)	ISO 10253; Marine algal growth inhibition test	S. costatum(marine)	72-h ErC50: 357

* Not determined (n.d.), due to a multiple sequentially-rejective U-test after Bonferroni-Holm.

** TTC (Toxic Threshold Concentration); comparable to ErC3

*** The ErC50 could not be calculated due to the limited number of replicates. However, as the highest concentration tested was 100 mg/L and the ErC10 is already calculated to be > 100 mg/L, the ErC50 is concluded to be clearly > 100 mg/L.

 

Key study:

The most sensitive result was obtained in a GLP study following the US EPA algal assay bottle test 600/9-78-018 (1978) and using the green algae Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum). The applied calculation method for the specific growth rates by using cell count values is comparable to current guidelines, e.g. OECD 201. Therefore, the reported EC50 for cell count can be regarded as related to the internationally preferred endpoint growth rate (ErC50). From the results of two separate tests, the mean 96-h ErC50 was 2.2 mg/l (OECD SIDS, 2007; Dow Chemical, 1982b; Cowgill et al., 1989).

Test 1 (started on 04.05.1981) and test 2 (started on 08.01.1982) were re-evaluated by using ToxRat Professional v2.10.

Test 1: The 72-h ErC10 was recalculated to be 1.4 mg/L (72-h EyC10: 0.7 mg/L); the 72-h ErC50 was determined to be 9.5 mg/L (96-h EyC50: 2.7 mg/L). The 96-h ErC10 was recalculated to be 1.4 mg/L (96-h EyC10: 0.5 mg/L) and the 96-h ErC50 was determined to be 9.7 mg/L (96-h EyC50: 2.0 mg/L). Due to a multiple sequentially-rejective U-test after Bonferroni-Holm, no 96-h LOErC and 96-h NOErC values could be calculated, but all the other NOEyC and LOEyC values for 72-h and 96-h as well as the NOErC and LOErC values for 72-h were calculated to be comparable: NOExC: 0.6 mg/L; LOExC: 1.0 mg/L.

Test 2: The 72-h ErC10 was recalculated to be 1.1 mg/L (72-h EyC10: n.d.); the 72-h ErC50 was determined to be 19 mg/L (96-h EyC50: 3.3 mg/L). The 96-h ErC10 was recalculated to be 1.4 mg/L (96-h EyC10: n.d.) and the 96-h ErC50 was determined to be 20.5 mg/L (96-h EyC50: 2.2 mg/L).

Both studies were selected as key study.

Remark on assessment of toxicity to algae:

The classification by long-term toxicity results is based on the lowest 72-h ErC10 of 1.1 mg/L determined in the key study for algae and not on the 72-h NOErC of 0.6 mg/L (recalculated with ToxRat v2.10). According to the Guidance on information requirements and chemical safety assessment Chapter R.10: Characterisation of dose [concentration]-response for environment ", an EC10 for a long-term test which is obtained using an appropriate statistical method (usually regression analysis) will be used preferentially. [...] There has been a recommendation within OECD in 1996 to phase out the use of the NOEC, in particular as it can correspond to large and potentially biologically important magnitudes of effect. The advantage of regression method for the estimation of ECx is that information from the whole concentration-effect relationship is taken into account and that confidence intervals can be calculated. These methods result in an ECx, where x is a low effect percentile (e.g. 5-20%). It makes results from different experiments more comparable than NOECs".

 

Supporting freshwater studies:

There are several supporting studies on freshwater algae available, indicating the ErC50 value of the key study (96-h ErC50= 9.7 mg/L; 72-h ErC50= 9.5 mg/L; P. subcapitata) to come up as uniquely low:

The toxicity of DEA to the same species, Pseudokirchneriella subcapitata, was examined in a study by the National Association of Photographic Manufacturers (NAPM; 1974) according to the U.S. EPA provisional algal assay procedure (PAAP). The 7-d ErC50 was determined to be in a range between 10 and 100 mg/L; the 7-d NOErC was 10 mg/L. In a cell-multiplication-inhibition test with Chlorella vulgaris according to a modification of an unspecified testing guideline, Zurita et al. (2005) determined the 72-h ErC50 to be 778 mg/L. Further studies according to the growth-inhibition-test method according to Bringmann were conducted with two freshwater algae species. Bringmann & Kühn (1977-1980) reported in several studies the Toxic Threshold Concentration(TTC, which is comparable to ErC3). For Microcystis aeruginosa an 8-d ErC3 of 16 mg/L was determined in neutralised test solutions. In non-neutralised test solutions the 8-d ErC3 was 17 mg/L. For Scenedesmus quadricauda an 8-d ErC3 of 4.4 mg/L was determined in neutralised test solutions. In non-neutralised test solutions the 8-d ErC3 was 10 mg/L.

 

Screening Tests:

The effect of neutralised and non-neutralised test solutions with DEA was mainly examined in screening studies with Ankistrodesmus bibraianus and Desmodesmus subspicatus in 72-hour static tests following the German Industrial Standard DIN 38412, part 9. These screening tests performed by BASF were selected as supporting studies. The following nominal test substance concentrations were set up in each of the four tests: 0, 0.78, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0, and 100.0 mg/l. Test concentrations were not verified analytically. The pH values were measured in all concentrations including control using a separate sample without algae at the beginning and the end (72 h) of the test. At the end of the test, pH measurements were also done in the inoculated samples. With neutralised samples, the pH values ranged from 7.7 - 8.1 at test start to 8.0 - 8.5 (without algae), respective 8.3 - 9.0 (with algae) after 72 h. In the non-neutralised samples, the pH values ranged from 7.4 - 8.8 at test start to 7.9 - 8.5 (without algae), respective 8.3 - 9.0 (with algae) after 72 h. The algal biomass was determined with a fluorimeter at the onset of the test and after 48 and 72 h.

The measured data of the screening tests were re-evaluated with ToxRat v2.10 (BASF SE, 2015). For Ankistrodesmus bibraianus the 72-h ErC50 under non-neutralised conditions was determined to be 57.5 mg/L (ErC10, non-neutralised: 30.1 mg/L). The ErC50 for neutralised test solutions could not be calculated due to the limited number of replicates. However, as the highest concentration tested was 100 mg/L and the chronic value for neutralised test solutions is already calculated to be > 100 mg/L (ErC10, neutralised: 123.9), the ErC50 under neutralised conditions is concluded to be clearly >100 mg/L. The respective 72-h ErC50 values for Desmodesmus subspicatus were 14.9 mg/l (neutralised) and 107.3 mg/l in non-neutralised test solutions (ErC10, neutralised: 2.4 mg/L; ErC10, non-neutralised: 85.7 mg/L).

With respect to potential pH effects, these results seem to be contradictory since in one case higher sensitivity was observed under non-neutralized compared to neutralized conditions, and in the other case this was just the opposite. However, pH neutralisation was carried out in these studies to correct the pH variation associated with  test substance, whereas the pH increases observed in all these tests were due to reduction in dissolved carbon dioxide in the media as the tests progressed (algal photosynthesis). It is likely that this continuing reduction in dissolved CO2 had a far greater effect on pH than preceding neutralisation for test substance. Hence, these differences in test results might also be due to intra-laboratory and/or biological variation (SIDS Initial Report for SIAM 24; Paris, France 17. - 20. April 2007).

 

Marine studies: 

From the representative data for marine algae reliable ErC10 values for chronic effects could not be deduced. In a study by Libralato et al. (2010) marine algae were tested according to ISO 10253. The test species was Phaeodactylum tricornutum. The concentration of the test item was analytically verified in the stock solution. Five test concentrations in a geometric series with a factor of 2 plus one control were used to determine the effect of DEA on marine algae. After 72 h of exposure an EC50 of 86.96 mg/L and a NOEC of < 16 mg/L were determined. Cowgill et al. (1988) tested the marine species Skeletonema costatum according to the U.S. EPA marine algal assay procedure, bottle test (1974). The test concentrations are not specified, but it was mentioned that ≥ 5 nominal concentrations have been used for testing. As the applied calculation method for the specific growth rates by using cell count values is comparable to current guidelines, e.g. OECD 201, the reported NOEC and EC50 for cell count can be regarded as related to the internationally preferred endpoint growth rate (NOErC and ErC50). A 5-d NOErC of 216 mg/L and a 5-d ErC50 of 548.2 mg/L were determined. Additionally, Eide-Haugmo et al. (2012/2009) tested the marine species S. costatum according to ISO 10253 and determined a 72 -h ErC50 of 357 mg/L.