Sulphophthalic acid derivatives reaction products of fatty alcohol C12-C14 and sodium hydroxide

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:

From January 21th to January 25th

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:

2011

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.2, 0.5, 1, 2, 5, 10, and 20 µg/ml
- Sampling method: test samples were diluited with ultra-pure water for the HPLC anlysis. Control sample were directly measured.

Vehicle:

no

Details on test solutions:

OECD medium (dilution water)
Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment.
Separate stock solutions were first prepared in deionised water. The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations in the prepared algal growth medium.

Test item concentrations in the definitive test
Based on the results of the preliminary range-finding test five test concentrations in a geometric series with a spacing factor of 2.0 and a concurrent untreated control were used in the main test.
The following nominal concentrations were tested: 6.25, 12.5, 25, 50 and 100 mg/l.
Measured test item concentrations remained within ± 20 % of the nominal over the test period of 48 hours, therefore the biological results are based on the nominal concentrations.

Preparation of test solutions
Test solutions used in the test were prepared by mechanical dispersion without using of any solubilising agent. A test item solution was first prepared by dissolving an amount of 0.1000 g test item in 1000 ml dilution water resulting a nominal concentration of 100 mg/l. This solution was filtrated through a membrane filter (0.22 µm) to separate the possible micelles formed because of the surface activity of the test item. The further test item solutions were prepared by the appropriate dilution of this test item stock solution with a dilution factor of 2.0. Test solutions were prepared just before introduction of algae (start of the experiment).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain number: 61.81 SAG
- Source (laboratory, culture collection): SAG: collection of algal cultures, D-37073 Gottigen, Germany
- Method of cultivation: the preculture is intended to give an amount of algae suitable for the inoculation of test cultures. Pre-culture was prepared with OECD medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days.
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Culture temperature was checked at the beginning of the study and every 24 hours thereafter in a flask filled with water, in the climatic chamber. In addition temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was between 22.7 and 22.9 °C measured in the flask and in the range of 21.3-23.7 °C measured within the climate chamber.

pH:

The pH was measured in each test group at the start (before test solutions had been distribuited into the test vessels) and in each test vessel at the end of the test. The pH of the control medium was not increased by more than 1.5 units during the test, in each vessel. The range of the pH was 7.63(min)-9.65(max) during the experiment. The pH of the test solutions was not adjusted during the study.

Nominal and measured concentrations:

Nominal concentrations: 6.25, 12.5, 25, 50, 100 mg/l
Measured concentrations t 0: 5.61±0.01, 11.3±0.05, 22.6±0.11, 45.4±0.83, 92.4±1.33 mg/l
Measured concentrations t 72h: 5.88±0.14, 12.0±0.03, 24.0±0.46, 47.9±1.64, 97.3±3.85 mg/l

Details on test conditions:

TEST SYSTEM
- Type: test flasks were covered with air-permeable stoppers
- Aeration: yes
- Initial cells density: 10(4) cells/ml
- No. of vessels per concentration (replicates): 3 replicates per test concentrations
- No. of vessels per control (replicates): 6 replicates in control group

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: 7832 lux which was ensured with fluorescent lamps. The differences in light intensity between the test vessels did not exceed ±15 % and therefore provided equal conditions for each test vessel.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

13.093 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

16.107 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

23.939 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

8.82 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13.725 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

95 % conf. limits
Growth rate:
EC10:10.385-15.378 mg/l
EC20: 13.414-18.497 mg/l
EC50: 21.022-27.416 mg/l
Yield:
EC10: 6.404-7.559 mg/l
EC20: 8.214-9.395 mg/l
EC50: 13.035-14.447 mg/l

Morphological changes of algal cells
No abnormal shape of algal cells was noticed in the control and at the test item concentration of 6.25 mg/l during the study. In the test group of 12.5 mg test item/l thin and swollen cells were found between normal cells. At the test item concentration of 25 mg/l the cells were swollen, while also thin cells appeared after 72 hours. At both of the highest test concentrations of 50 and 100 mg/l swollen cells were found during the experiment.

Validity of the test
The cell density in the control cultures increased by a factor of 140.50 within 72 hours. This corresponds to a specific growth rate of 1.65 day-1.
The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %
- CV for section-by-section growth rate day 0-1: 10.24 %
- CV for section-by-section growth rate day 1-2: 4.04 %
- CV for section-by-section growth rate day 2-3: 3.05 %
The mean coefficient of variation for section-by-section specific growth rates: 5.78 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.61 %.
All validity criteria were met, therefore the study can be considered as valid.

Growth rates (µ) and percentage inhibition of µ during the test period

concentration [mg/l]	growth rates (µ) and % inhibition of µ
	0–24 h		0–48 h		0–72 h
	µ	%	µ	%	µ	%
control	0.0478	-	0.0671	-	0.0686	-
6.25	0.0401	16.0	0.0653	2.8	0.0675	1.7
12.5	0.0193	59.7	0.0574	14.5	0.0615	10.3
25	0.0000	100.0	0.0096	85.7	0.0410	40.2
50	0.0000	100.0	0.0000	100.0	0.0000	100.0
100	0.0000	100.0	0.0000	100.0	0.0000	100.0

 

Yield (Y) and percentage inhibition of Y during the test period

concentration [mg/l]	yield (y) and % inhibition of y
	0–72 h
	y	%
control	139.50	-
6.25	128.00	8.2
12.5	83.33	40.3
25	18.33	86.9
50	0.00	100.0
100	0.00	100.0

 

Validity criteria fulfilled:

yes

Conclusions:

Growth rate, EC50 (72h) = 23.939 mg/l.
Yield, EC50 (72h) = 13.725 mg/l.
For both, NOEC (72h) = 6.25 mg/l, LOEC (72h) = 12.5 mg/l.

Executive summary:

Toxicity to acquatic plants was detrmined following OECD guideline 201.

Exponentially-growing algal cultures were tested in OECD medium, at nominal concentrations of 0, 6.25, 12.5, 25, 50, 100 mg/l. Measured concentrations remained within ±20 % of the nominal over the test period, thus biological results are based on the nominal concentrations.

Algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10⁴ cells/ml at the start of the test in all of the test cultures. Alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24 -hour intervals.

Analytical monitoring was done by HPLC method with UV detection.

Effect levels in terms of EC10, EC20, EC50, NOEC and LOEC for both response variables (i.e. average specific growth rate and yield) were determined.

Statistics: for the detemination of the LOEC and NOEC, Jonckheere-Terpstra Test, for determination of the ECx values Probit analysis was used (statistical software program SPSS PC+).

All validity criteria were met.

Effect levels after a 72 h exposure were:

- for growth rate, EC50 = 23.939 mg/l

- for yield, EC50 = 13.725 mg/l

- for growth rate and yield, NOEC = 6.25 mg/l and LOEC = 12.5 mg/l.

Description of key information

Effect levels after a 72 h exposure were:

- for growth rate, EC50 = 23.939 mg/l

- for yield, EC50 = 13.725 mg/l

- for growth rate and yield, NOEC = 6.25 mg/l and LOEC = 12.5 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

23.939 mg/L

EC10 or NOEC for freshwater algae:

6.25 mg/L

Additional information

Test conducted according to OECD guideline 201.

Nominal concentrations of 0, 6.25, 12.5, 25, 50, 100 mg/l were investigated in the main study. Measured concentrations remained within ±20 % of the nominal over the test period of 72 hours.

The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10⁴cells/ml at the start of the test in all of the test cultures. Alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24 -hour intervals.

Effect levels were determined for both growth rate and yield. All validity criteria were met.