2-(2-aminoethylamino)ethanol

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2020 - 16 Jun 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-aminoethylamino)ethanol

EC Number:

203-867-5

EC Name:

2-(2-aminoethylamino)ethanol

Cas Number:

111-41-1

Molecular formula:

C4H12N2O

IUPAC Name:

2-[(2-aminoethyl)amino]ethan-1-ol

Test material form:

liquid: viscous

Details on test material:

Physical Description: Colourless viscous liquid

Purity/Composition correction factor: No correction factor require

Storage Conditions: At room temperature protected from light container flushed with nitrogen

Test item handling: Use amber glassware or wrap container in aluminum foil

Specific details on test material used for the study:

The stability of the test substance was determined using Infra Red (CRL study no. 20230572). For the acute algae study an infrared absorption spectrum was obtained at the start, during and at the end of the study. All infrared absorption spectra were comparable. From this it could be concluded that the test item was stable during the acute algae study.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 h and t=72 h.
Volume: 2.0 mL from the approximate centre of the test solutions.
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 3.2 mg/L but without algae (abiotic control) and samples for analysis were taken at the start and at the end of the test period.
Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Preparation of test solutions started with the highest concentration of 100 mg/L applying 15 minutes of magnetic stirring to accelerate dissolution of the test item in medium. In the range-finding test, the pH of this solution was adjusted from 9.7 to 8.3 using 1M HCL-solution (Merck, Darmstadt, Germany) to lay within the optimal pH-range for algal growth, specified in the OECD Test Guideline 201 (i.e. between 6 and 9). In the final test, pH was adjusted from 9.9 to 8.5. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The pH of blank medium used for the control group and dilutions was adjusted from 8.1 to 8.6 in the range-finding test and to 8.5 in the final test using 1M NaOH-solution (Merck, Darmstadt, Germany). All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Species: Raphidocelis subcapitata, strain: NIVA CHL 1
- Source: In-house laboratory culture.
- Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

CULTURE
- Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Pre-culture: Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x E4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

between 22 and 24°C

pH:

Between 8.0 and 8.5

Nominal and measured concentrations:

Nominal concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.
Measured concentrations: The measured concentrations were at the level of nominal throughout the test, i.e. were in the range of 88 to 115% of the nominal values at the start and the end of the exposure period. Based on the obtained results, effect parameters were expressed in terms of analytically confirmed nominal concentrations.

It should be noted that small test item responses were detected in the control samples taken at the start and the end of the test. The response detected in the control solution at test commencement was comparable to the response detected in the blank Quality Control (QC) samples, and therefore, was probably introduced during sample pre-treatment. The response measured in the sample taken at the end of the test was slightly higher, yet considered negligible compared to the lowest test concentration.
The concentrations measured in the samples taken from solution without algae (abiotic control) were comparable with the concentrations measured in the samples with algae. Hence, it can be stated that the presence of the algae did not affect the concentration of the test item in test medium throughout the test.

Details on test conditions:

TEST SYSTEM
- Test vessels: 100 mL, all-glass with aluminum caps, perforated for ventilation, containing 50 mL of test solution.
- Test Medium: M2
- Cell density: An initial cell density of 1 x E4 cells/mL.
- Illumination: Continuously using TLD-lamps with a light intensity of 87 µE.m-2.s-1.
- Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Control: Test medium without test item or other additives.
- Replicates: 3 replicates of each test concentration, 6 replicates of the control, 1 or 2 replicates of each test concentration without algae.
-pH adjustment: yes

TEST MEDIUM / WATER PARAMETERS
M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl: 15 mg/L
MgCl2.6H2O: 12 mg/L
CaCl2.2H2O: 18 mg/L
MgSO4.7H2O: 15 mg/L
KH2PO4: 1.6 mg/L
FeCl3.6H2O: 64 µg/L
Na2EDTA.2H2O: 100 µg/L
H3BO3: 185 µg/L
MnCl2.4H2O: 415 µg/L
ZnCl2: 3 µg/L
CoCl2.6H2O: 1.5 µg/L
CuCl2.2H2O: 0.01 µg/L
Na2MoO4.2H2O: 7 µg/L
NaHCO3: 50 mg/L
Hardness (Ca+Mg): 0.24 mmol/L (24 mg CaCO3/L)
pH :8.1 ± 0.2

EFFECT PARAMETERS MEASURED

- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank.

TEST CONCENTRATIONS
- Range finding study: a rangefinding study was performed with the concentrations of 0.1, 1.0, 10, and 100 mg/L.
- Results used to determine the conditions for the definitive study: yes
- Final study:
- Spacing factor for test concentrations: 3.2
- Test concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 24 -90 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Inhibition of growth rate and yield increased with increasing concentration of the test item, resulting in 13% growth rate and 52% yield inhibition at the highest concentration tested. Statistically significant growth rate and yield inhibition was found at all test concentrations.
Due to the shallowness of the dose-response curve and limitations to the test design (i.e. maximum spacing factor and number of test groups), the NOEC for growth rate and yield inhibition could not be determined. Instead, the EC10 for growth rate was calculated as an equivalent alternative.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest test concentration when compared to the control.

Reported statistics and error estimates:

For determination of the NOEC and the ECx the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test, α=0.05, one-sided, smaller).

Calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding concentrations of the test item. Since the slope of the obtained dose-response curve was shallow, the ERC10 (growth rate inhibition) and the EYC50 (yield inhibition) were determined using the three highest test concentrations only in order to improve model-fit.

The ERC20 and ERC50 could not be determined because the observed effects were below 20%. The EYC10 and EYC20 were both below the range of concentrations tested. However, the EYC20 could be estimated by extrapolation of the modelled dose-response curve using all test groups.

Any other information on results incl. tables

Table 1: Growth Rate and Percentage Inhibition for the Total Test Period       

2-(2-aminoethylamino)ethanol Nominal concentration (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.937	0.0237	6
0.32	1.855	0.0477	3	4.2*
1.0	1.825	0.0547	3	5.8*
3.2	1.803	0.0958	3	6.9*
10	1.796	0.0315	3	7.3*
32	1.782	0.0225	3	8.0*
100	1.692	0.0477	3	13*

* Effect was statistically significant

Table 2:        
Growth Rate and Percentage Inhibition at Different Time Intervals

Test item[1] Nominal conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.366		1.899		1.547
0.32	3	2.428	-2.6	1.644	13	1.493	3.5
1.0	3	2.432	-2.8	1.592	16	1.450	6.3
3.2	3	2.379	-0.57	1.632	14	1.398	9.7
10	3	2.417	-2.2	1.513	20	1.459	5.7
32	3	2.386	-0.87	1.591	16	1.367	12
100	3	2.347	0.81	1.556	18	1.175	24

Table 3          
Yield and Percentage Inhibition for the Total Test Period

2-(2-aminoethylamino)ethanol Nominal concentration (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	334.1	23.78	6
0.32	262.0	37.02	3	22*
1.0	239.8	40.45	3	28*
3.2	228.2	60.94	3	32*
10	218.7	21.26	3	35*
32	208.8	14.09	3	37*
100	160.5	23.84	3	52*

* Effect was statistically significant.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See"overall remarks"

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, 2-(2-aminoethylamino)ethanol inhibited growth rate of this freshwater algae species significantly at analytically confirmed nominal concentrations of 0.32 mg/L and higher.
The 72h-EC10 for growth rate inhibition (ERC10) was 46 mg/L with a 95% confidence interval ranging from 24 to 90 mg/L. The 72-hour EC20 and EC50 for growth rate inhibition (ERC20 and ERC50) were beyond the range of concentrations tested, i.e. exceeded the regulatory limit concentration of 100 mg/L.

The 72h-EC50 for yield inhibition (EYC50) was 98 mg/L with a 95% confidence interval ranging from 62 to 245 mg/L. The 72h-EC10 for yield inhibition (EYC10) was below the range of concentrations tested, and therefore, could not be determined reliably. Contrary, the EYC20 was estimated to be at 0.20 mg/L, with a 95% confidence interval ranging from 0.010 to 0.77 mg/L, by extrapolation of the modelled dose-response curve.
The NOEC for both growth rate and yield inhibition could not be determined due to the flatness of the dose-response curve and limitations to the test design (maximal spacing factor and number of test groups).

Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles with 2-(2-aminoethylamino)ethanol, freshwater algae (Pseudokirchneriella subcapitata) were exposed to 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. The measured concentrations were at the level of nominal throughout the test, i.e. were in the range of 88 to 115% of the nominal values at the start and the end of the exposure period. Based on the obtained results, effect parameters were expressed in terms of analytically confirmed nominal concentrations. Inhibition of growth rate increased with increasing concentration of the test item, resulting in 13% growth rate inhibition at the highest concentration tested. Statistically significant growth rate inhibition was found at all test concentrations.

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, 2‑(2‑aminoethylamino)ethanol inhibited growth rate of this freshwater algae species significantly at analytically confirmed nominal concentrations of 0.32 mg/L and higher. The 72h-EC₁₀ for growth rate inhibition (E_RC₁₀) was 46 mg/L with a 95% confidence interval ranging from 24 to 90 mg/L. The 72-hour EC₂₀ and EC₅₀ for growth rate inhibition (E_RC₂₀and E_RC₅₀) were beyond the range of concentrations tested, i.e. exceeded the regulatory limit concentration of 100 mg/L. The study met all validity criteria, and is considered reliable without restriction.