Tris(2-hydroxyethyl)ammonium salts of tall-oil fatty acids

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

REG (EC) No. 440/2008, May 2008 amended by REG (EC) No. 761/2009, July 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal 100 , 50, 25, 12.5, 6.25 mg/L as WAF and control
- Sampling intervals: start and end of the test (0 and 72 h)
- Sampling method: samples were taken from flasks containing algal cells
- Sample storage conditions before analysis: processed (freeze-dried) samples were analysed on the day of sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAFs)
Supersaturated solutions were prepared by dispersing/dissolving the test item into the test medium (OECD Medium) at nominal concentrations of 100, 50, 25 and 12.5 mg/L. These dispersions were shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at the test temperature. The resulting dispersions were opalescent. The non-dissolved test material was removed by filtration through a 0.22-µm filter to provide the WAFs. The filtrates were clear and transparent. The lowest nominal test concentration of 6.25 mg/L was prepared by diluting the 100 mg/L WAF.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, GERMANY.
- Age of inoculum (at test initiation): Three days. Algal cells used for inoculation were in the exponential phase of growth.
- Method of cultivation: Algae are cultured under standardised conditions. The pre-culture was intended to give an amount of alga suspension suitable for the inoculation of test cultures. The pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal Growth Medium to 10^7 cells/mL.

ACCLIMATION
- Acclimation period: 3 days (pre-culturing)
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L as CaCO3 (calulated)

Test temperature:

22.5-22.8 °C (daily measurements in a flask filled with water and incubated under the test conditions)
22.1-23.3 °C (continuous measurements in the climatic chamber)

pH:

test start (0 h): 7.8-8.1
test end (72 h): 8.0-9.3
The pH did not increase by more than 1.5 units in the control and in the treatments

Nominal and measured concentrations:

nominal concentrations: 100, 50, 25, 12.5, and 6.25 mg/L as WAF
measured concentrations:
start (0h): 10.6, 9.0, 3.9, 2.4, 0.69 mg/L
end (72 h): 1.0, 0.73, 0.30, 0.16, 0.12 mg/L
geometric mean measured concentrations: 3.3, 2.6, 1.1, 0.6 and 0.3 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks containing 100 mL of inoculated test medium and covered with air-permeable stoppers
- Aeration/gaseous exchange: continuous shaking on an laboratory orbital shaker
- Initial cells density: 1x10⁴ cells/mL
- Control end cells density: 76 x10⁴ cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Ca/Mg ratio: 1:1
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: about 8116 lux (equivalent to 110 µE/m2/s), provided by fluorescent lamps (spectral range 400-700 nm).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): cell numbers counted at 24, 48 and 72 hours
- Determination of cell concentrations: counting chamber
- Algal cells were observed microscopically for abnormal appearance at 24, 48 and 72 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study
- Test concentrations: nominal 100, 10, 1 mg/L (WAFs) and 0.1 mg/L (dilution of 1 mg/L)
- Results used to determine the conditions for the definitive study: inhibition of algal growth (yield) after 72 hours was >95% at nominal loading rates of 100 and 10 mg/L; no significant inhibition was observed at the two lower concentrations (≤ 7%)

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 21-26 mg/L; results are expressed in terms of loading rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks:

(yield)

Remarks on result:

other: 95% confidence limits: 12-14 mg/L; results are expressed in terms of loading rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits: 13-16 mg/L; results are expressed in terms of loading rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Remarks on result:

other: results are expressed in terms of loading rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Remarks on result:

other: results are expressed in terms of loading rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no microscopic abnormalities
- Any stimulation of growth found in any treatment: no

Control cultures:
- increase of biomass by a factor of: 76, corresponding to a specific growth rate of 1.44 per day
- mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3): 16%
- coefficient of variation for the average specific growth rates during the test period (day 0-3): 0.60%

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72-hour ErC50: 0.94 mg/L; 95% confidence limits: 0.85-1.03 mg/L;
- 72-hour EbC50: 0.54 mg/L; 95% confidence limits: 0.49-0.59 mg/L.
These values are within the range of the laboratory ring test data (ISO Guideline No. 8692).

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) to demonstrate exponential growth for the entire study period.

The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).

The EL50 values of the test item for growth (r), biomass (b) and yield (y) were determined using Probit analysis by TOXSTAT software.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.

For the determination of the LOELR and NOEL, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Table 1: Cell Number

Nominal loading rates (mg/L)	Replicate number	Cell number (x 10^4/mL)
		24 hours	48 hours	72 hours
Control	R1	4	20	78
	R2	6	20	76
	R3	5	19	74
	R4	6	18	78
	R5	5	20	74
	R6	6	21	78
	Mean	5.33	19.67	76.33
	SD	0.8	1.0	2.0
6.25	R1	6	20	76
	R2	6	17	70
	R3	4	20	77
	Mean	5.33	19.00	74.33
	SD	1.2	1.7	3.8
12.5	R1	2	12	21
	R2	3	13	31
	R3	2	12	21
	Mean	2.33	12.33	24.33
	SD	0.6	0.6	5.8
25	R1	2	12	20
	R2	1	11	14
	R3	2	12	20
	Mean	1.67	11.67	18.00
	SD	0.6	0.6	3.5
50	R1	2	2	2
	R2	1	1	1
	R3	1	1	1
	Mean	1.33	1.33	1.33
	SD	0.6	0.6	0.6
100	R1	1	1	1
	R2	1	1	1
	R3	1	1	1
	Mean	1.00	1.00	1.00
	SD	0.0	0.0	0.0

R1-R6: replicate number

Note: the initial cell density was estimated to be 1 x 10⁴/mL

 

 

Table 2  Inhibition of growth

Parameter	Nominal loading rate (mg/L)	Sample size	Mean	Inhibition (%)
Area under curve 0 to 72 hours	Control	6	1456	N/A
	6.25	3	1416	2.7
	12.5	3	584*	59.9
	25	3	476*	67.3
	50	3	20*	98.6
	100	3	0.0*	100.0
Growth rate 0 to 24 hours	Control	6	0.0693	N/A
	6.25	3	0.0690	0.4
	12.5	3	0.0345*	50.2
	25	3	0.0193*	72.2
	50	3	0.0096*	86.1
	100	3	0.0000*	100.0
Growth rate 0 to 48 hours	Control	6	0.0620	N/A
	6.25	3	0.0613	1.2
	12.5	3	0.0523*	15.7
	25	3	0.0512*	17.5
	50	3	0.0048*	92.2
	100	3	0.0000*	100.0
Growth rate 0 to 72 hours	Control	6	0.0602	N/A
	6.25	3	0.0598	0.6
	12.5	3	0.0441*	26.8
	25	3	0.0400*	33.6
	50	3	0.0032*	94.7
	100	3	0.0000*	100.0
Yield	Control	6	75.3	N/A
	6.25	3	73.3	2.7
	12.5	3	23.3*	69.0
	25	3	17.0*	77.4
	50	3	0.3*	99.6
	100	3	0.0*	100.0

* : statistically significantly different compared to the control values (Bonferroni t-Test;α= 0.05).

 

Validity criteria fulfilled:

yes

Remarks:

Increase of biomass >16 after 72 hours; mean coefficient of variation for section-by-section specific growth rate <35%; coefficient of variation for average specific growth rate <7%.

Conclusions:

The test material is toxic to freshwater algae with a 72-hour ErL50 between 10 and 100 mg/L (calculated value: 23 mg/L).

Description of key information

effect values for average specific growth rates of Pseudokirchneriella subcapitata, 0-72 h (based on nominal loading rates) (OECD 201, EU C.3):
EC50: 23 mg/L, NOEC: 12.5 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

23 mg/L

EC10 or NOEC for freshwater algae:

12.5 mg/L