Tributylethylammonium ethyl sulphate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2017 - 03 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28 July 2011

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

A correction factor of 0.368 was applied for the the concentration of the anion (36.8%).

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 and t=72
Volume: 2.0 mL
Storage: Samples were stored in a freezer (≤ 15°C) until analysis.

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 the limit concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: Direct application to the test medium: preparation of test solutions started with the highest concentration (range-finding test: 100 mg/L (37 mg anion/L); final test: 272 mg/L (100 mg anion/L)). Since the test item was completely soluble in test medium, no other treatment than vigorous shaking was needed to completely dissolve the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Evidence of undissolved material: All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture

CULTIVATION:
- Method: 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/m^2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm
- Medium different from test medium: yes, M1

ACCLIMATION
- Acclimation period: 3 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 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Acclimation medium different from test medium: no, M2 (according to OECD 201)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

22 - 23°C throughout the test.

pH:

Start: 8.1-8.2
End: 7.6-7.9

Nominal and measured concentrations:

Nominal concentrations: 0.39, 1.6, 6.3, 25 and 100 mg anion/L corresponding to 1.1, 4.3, 17, 68 and 272 mg test substance/L.
Measured concentrations at t=0: 1.17, 4.7, 17.8, 69, 73 and 294 mg test substance/L based on the anion concentration and 1.42, 4.71, 18.8, 72.5, 75.0 and 301 mg test substance/L based on cation concentration
Concentrations at t=72 were 101-104% of initial in concentrations based on the anion concentration and 85-104% of initial in concentrations based on the cation concentration. Based on these results the effect concentrations were based on the nominal concentrations mg test substance/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, open, fill volume: 50 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 198.9 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Other: 1 or 2 extra replicates of each test concentration without algae

TEST MEDIUM / WATER PARAMETERS
- Standard test medium used: yes, M2 according to OECD 201
- Source of dilution water: Milli-RO water
- Culture medium different from test medium: yes, M1

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: continuous illumination using TLD-lamps with a light intensity within the range of 81 to 84 µE/m^2/s

EFFECT PARAMETERS MEASURED: growth rate at 72 hours.
- Additional measurements: pH at the beginning and at the end of the test. Temperature: continuously in a temperature control vessel; Appearance of the cells: at the end of the final test microscopic observations were performed on the intermediate test concentration (4.3 mg/L) to observe for any abnormal appearance of the algae.
- 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 (pathlength =20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.

COMBINED LIMIT/ RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 mg test item/L (0.037, 0.37, 3.7 and 37 mg anion/L)
- Results used to determine the conditions for the definitive study: yes, growth rate was inhibited for 33% in the highest tested concentration. The expected EC50 for the final test was >100 mg test item/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (June 2017)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.6 mg/L

Nominal / measured:

nominal

Conc. based on:

other: anion

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.82 mg/L

Nominal / measured:

nominal

Conc. based on:

other: anion

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.39 mg/L

Nominal / measured:

nominal

Conc. based on:

other: anion

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: cation

Basis for effect:

growth rate

Remarks on result:

other: cation concentration calculated

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.4 mg/L

Nominal / measured:

nominal

Conc. based on:

other: cation

Basis for effect:

growth rate

Remarks on result:

other: cation concentration calculated

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.7 mg/L

Nominal / measured:

nominal

Conc. based on:

other: cation

Basis for effect:

growth rate

Remarks on result:

other: cation concentration calculated

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Effect parameters were expressed in terms of analytically confirmed nominal concentrations of the anion and calculated to mg test item/L. Analysis showed that measured concentrations of the anion were 102-112% of nominal concentrations. Since there was an outlier in measured concentrations of the cation while all remaining concentrations were at the level of nominal concentrations, it was justified to base the effect concentrations on nominal concentrations of the anion.
- Effect parameters based on cation were calculated by data submitter, using the nominal effect concentrations for the test item in combination with the correction factor for the cation.
- Test conditions (pH and temperature) remained within the limits prescribed by the study plan.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Concentrations tested: 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- 72h-ErC50: 0.99 mg/L (95% CI: 0.97-1.0 mg/L)
- Other: the result fell within the historical data range (0.82 - 2.3 mg/L).

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, 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 TWA concentrations of the test item.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1 Measured concentrations (anion) versus nominal concentrations

Time of sampling [hours]	Date of sampling	Date of analysis^a	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
			Nominal	Analyzed
0	17 Jul 2017	02 Aug 2017	0	n.d.	n.a.
			1.1	1.17	107
			4.3	4.7^c,d	108
			17	17.8	105
			68	69^c,d	102
			68^b	73^c,d	107
			272	294	108
72	20 Jul 2017	02 Aug 2017	0	0.0216	n.a.
			1.1	1.22	111	104
			4.3	4.8^c,d	112	103
			17	18.0	106	101
			68	71^c,d	104	103
			68^b	74^c,d	109	102
			272	295	109	101

^a      Samples were stored in the freezer (≤ -15°C) until the day of analysis.; ^b      Without algae.; ^c      Estimated value, calculated by extrapolation of the calibration curve.; ^d         Since cation results where acceptable (the same results with the curve with broader range) and not extrapolated, anion results are accepted without additional analysis.

n.a.Not applicable.

Table 2 Growth rate and percentage inhibition for the total test period

TBEAES DRY Nominal conc. (mg anion/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.762	0.0383	6
0.39 (1.1)	1.745	0.0186	3	1.0
1.6 (4.3)	1.338	0.0635	3	24*
6.3 (17)	0.534	0.1360	3	70*
25 (68)	0.125	0.1675	3	93*
100 (272)	0.055	0.0705	3	97*

* effect was statistically significant; () between brackets, the concentration of the test item is given (mg/L)

Validity criteria fulfilled:

yes

Remarks:

see 'overall remarks'

Conclusions:

The ErC50, ErC10 and NOErC of TBEAES DRY were 9.7, 2.2 and 1.1 mg/L, respectively, based on the nominal concentration of the test substance in test medium (calculated from analytically confirmed nominal concentrations of the anion in test medium).

Executive summary:

A study was performed to assess the effect of TBEAES DRY on the growth rate of fresh water algae (Pseudokirchneriella subcapitata) after 72 hours of exposure. The study was conducted in accordance with OECD 201 and GLP.

 

Test solutions were prepared by adding the test item directly to the test medium and shaking the solutions vigorously for complete dissolution of the test item. A combined limit/range-finding study was performed with test solutions prepared individually at loading rates of 0.1, 1.0, 10 and 100 mg test item/L.

 

In the final test three replicates of exponentially growing algal cultures with an initial algal cell density of 10^4cells/mL were exposed to nominal concentrations of 1.1, 4.3, 17, 68 and 272 mg/L (0.39, 1.6, 6.3, 25 and 100 mg anion/L). Six replicates of a blank control and one replicate per test item concentration without algae were included. Samples were taken from all test concentrations and the blank control and analysed for confirmation of actual exposure concentrations at the start and after 72 hours of exposure. Measured concentrations of the anion were 102-112% of nominal concentrations. Since the test item remained stable over the 72 hours exposure period, effect concentrations were based on the test item concentration calculated from the nominal concentrations of the anion.

A dose-related increase of inhibition of growth rate at the end of the test was observed. The 72h-ErC50 was 9.7 mg/L (3.6 mg anion/L), the 72h-ErC10 was 2.2 mg/L (0.82 mg anion/L), and the 72h-NOErC was 1.1 mg/L (0.39 mg anion/L). All acceptability criteria were met and the study was considered to be valid.

Description of key information

A study was performed to assess the effect of TBEAES DRY on the growth rate of fresh water algae (Pseudokirchneriella subcapitata) after 72 hours of exposure. The study was conducted in accordance with OECD 201 and GLP. Test solutions were prepared by adding the test item directly to the test medium and shaking the solutions vigorously for complete dissolution of the test item. A combined limit/range-finding study was performed with test solutions prepared individually at loading rates of 0.1, 1.0, 10 and 100 mg test item/L. In the final test three replicates of exponentially growing algal cultures with an initial algal cell density of 10^4 cells/mL were exposed to nominal concentrations of 1.1, 4.3, 17, 68 and 272 mg/L (0.39, 1.6, 6.3, 25 and 100 mg anion/L). Six replicates of a blank control and one replicate per test item concentration without algae were included. Samples were taken from all test concentrations and the blank control and analysed for confirmation of actual exposure concentrations at the start and after 72 hours of exposure. Measured concentrations of the anion were 102-112% of nominal concentrations. Since the test item remained stable over the 72 hours exposure period, effect concentrations were based on the test item concentration calculated from the nominal concentrations of the anion. A dose-related increase of inhibition of growth rate at the end of the test was observed. The 72h-ErC50 was 9.7 mg/L (3.6 mg anion/L), the 72h-ErC10 was 2.2 mg/L (0.82 mg anion/L), and the 72h-NOErC  was 1.1 mg/L (0.39 mg anion/L). All acceptability criteria were met and the study was considered to be valid.