Reaction mass of sodium hydrogen N,N-dibutyl-10-(sulphonatooxy)octadecanamidate and sodium sulphooctadecanoate

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:

2012-09-18 to 2012-09-21

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:

adopted March 23, 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

August 24, 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"

Version / remarks:

December 15, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

One sample from the freshly prepared stock solution and duplicate samples from the freshly prepared test media (without algae) of all test concentrations and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated during test period under the same conditions as in the actual test (but without algae) and from these test media duplicate samples were taken at the end of the test period.

Vehicle:

no

Details on test solutions:

The test item was not well soluble in test water. Therefore, a stock suspension of 100 mg test item/L was prepared by suspending 101.5 mg test item in 1015 mL test water for preparing the test concentrations. The stock suspension was stirred for 1 hour to dissolve as much test item as possible. Then, non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 µm cellulose nitrate filter). The solution with dissolved test item was used as the test medium of the highest test concentration and to prepare the desired 1:2, 1:4, 1:8 and 1:16 dilutions. The test media were prepared just before introduction of the algae.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Strain No. 61.81 SAG
- Source: "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", 37073 Göttingen, Germany.
- Breeding conditions: The algae were cultivated in the laboratories of IBACON under standardised conditions according to the test guidelines.

ACCLIMATION
- Culturing media and conditions: same as test conditions
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

23 °C

pH:

8.1 to 8.2 at test start and
7.5 to 9.0 at test end

Nominal and measured concentrations:

Nominal: 320, 101, 32, 10.1 and 3.2 mg test item/L and a control.
Measured: Within ± 20 % of the nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed
- Material, size, fill volume: glass, 50 mL, 50 mL
- Aeration: no
- Initial cells density: 5000 algae cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, reconstituted water prepared according to guideline

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: Reconstituted water (OECD Medium)

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: Continuous illumination
- Light intensity and quality: 6208 lux (range: 5780 to 6630 lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell densities in the samples were determined by spectrophotometric measurement.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.15
- Range finding study: yes
- Test concentrations: 320, 101, 32, 10.1 and 3.2 mg test item/L and a control
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

73 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 54.7- 98.1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % C.I: 14.9 - 20.6

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

21.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % C.I: 11.9 - 30.4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

7.11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% C.I: 5.30 - 8.79

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I: 4.95 - 17.8

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.44 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % C.I: 2.97 - 5.85

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

<= 3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The 72-hour ErC50 value was calculated to be 73.0 mg test item/L and the 72-hour EyC50 was calculated to be 17.5 mg test item/L. The 72-hour NOErC was determined to be 3.2 mg test item/L and the associated 72-hour LOErC is 10 mg test item/L. The 72-hour NOEyC was determined to be lower than 3.2 mg test item/L and the associated 72-hour LOEyC is at least 3.2 mg test item/L.

Reported statistics and error estimates:

Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis. For the determination of the 72-hour LOEC and the 72-hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by the Williams t-test (growth rate, yield), respectively. The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat® Solutions GmbH.

Biological Results

Table 1: Yield y and Percentage Inhibition of y during the Test Period

Nominal concentration [mg test item/L]	Yields y [10^4cells/mL] and % inhibition of y
	24 hours		48 hours		72 hours
	y	%	y	%	y	%
Control	1.524	-	15.739	-	95.018	-
3.2	1.524	0.0	14.549	7.6	85.409	10.1*
10	1.878	-23.2	14.236	9.5*	68.070	28.4*
32	2.055	-34.9	7.351	53.3*	27.632	70.9*
100	1.229	19.4	0.653	95.9*	1.779	98.1*
320	0.875	42.6*	0.840	94.7*	1.592	98.3*

negative values in `% inhibition` indicate an increase in growth relative to that of the control

* mean value significantly different from the control (tested with Williams t-test, a = 0.05, one-sided)

Table 2: Growth Rates µ and Percentage Inhibition of µ during the Test Period

	Growth rates µ [1/day] and % inhibition of µ
Nominalconcentration [mg test item/L]	0 -	24 hours	0 -	48 hours	0 -	72 hours
	µ	%	µ	%	µ	%
Control	1.396	-	1.740	-	1.751	-
3.2	1.397	-0.1	1.702	2.2	1.715	2.0
10	1.557	-11.5	1.688	3.0	1.636	6.5*
32	1.607	-15.1	1.376	20.9*	1.342	23.3*
100	1.226	12.2	0.408	76.6*	0.502	71.3*
320	0.998	28.5*	0.486	72.1*	0.468	73.3*

negative values in `% inhibition' indicate an increase in growth relative to that of the control

* mean value significantly different from the control (tested with Williams t-test, a = 0.05, one-sided)

Table 3: Analytical Results

Determination of the Test Item:	Based on the results of TOC measurements the concentration of the test item was determined using a calibration curve
Calibration Ranges:	TC: 2.5 to 100 mf Carbon/L IC: 1 to 25 mg Carbon/L
Linearity of Response:	Correlation of peak area of different standard solutions with their corresponding concentrations, using a linear regression
Regression Coefficient (r^2):	TC: at least 0.9987 IC: at least 0.9973
Typical Calibration Curves:	TC: y = 3.992 * x - 1.291 IC: y = 3.933 * x - 0.575
Limit of Detection:	TC: 8.894 mg Carbon/L IC: 1.991 mg Carbon/L
Limit of Quantification:	30 mg test item/L mean recovery rate: 98 % (n = 5; RSD 8 %)
Mean Recovery in the Fortified Samples:	97 % (n = 15, RSD 6 %)
Mean Recovery in the Test Samples:	After 0 h: 95 % (n = 6, RSD 6 %) After 72 h: 90 % (n = 6, RSD 7 %)

Table 4: Summary of Analytical Results

sample description [mg/L]	% of nominal^1	RSD	n
Control	< LOQ	n.a.	2
3.2	< LOQ	n.a.	4
10	< LOQ	n.a.	4
32	88	6	4
100	90	1	4
320	100	5	4

¹mean value of all measured samples per treatment group

RSD: relative standard deviation per treatment group

n: number of analysed samples

n.a.: not applicable

At the start of the test 95 % of the nominal test concentrations were found (average of all test concentrations of nominal 32 to 320 mg/L). After 72 hours test duration, 90 % of the nominal value was determined (average of all test concentrations of nominal 32 to 320 mg/L). The test item concentrations of nominal 3.2 and 10 mg/L were below the Limit of Detection or the Limit of Quantification. During the test the algae were exposed to a mean of 93 % of nominal. Therefore, all reported results refer to nominal concentrations.

Validity criteria fulfilled:

yes

Conclusions:

The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test. The 72-hour ErC50 value was calculated to be 73.0 mg test item/L and the 72-hour EyC50 was calculated to be 17.5 mg test item/L. The 72-hour ErC10 value was calculated to be 11 mg test item/L and the 72-hour EyC10 was calculated to be 4.44 mg test item/L. The 72-hour NOErC was determined to be 3.2 mg test item/L and the associated 72-hour LOErC is 10 mg test item/L. The 72-hour NOEyC was determined to be lower than 3.2 mg test item/L and the associated 72-hour LOEyC is at least 3.2 mg test item/L.

Executive summary:

Toxicity of the test item to Pseudokirchneriella subcapitata in an Algal Growth Inhibition Test determined according to OECD TG 201 and EU method C.3. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The study comprised of 6 treatment groups (5 dose rates of the test item, control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometric measurement. The samples of the test media sampled at the start and at the end of the test after 72 hours of exposure were analysed via TOC-method. At the start of the test 95 % of the nominal test concentrations were found (average of all test concentrations of nominal 32 to 320 mg/L). After 72 hours test duration, 90 % of the nominal value was determined (average of all test concentrations of nominal 32 to 320 mg/L). The test item concentrations of nominal 3.2 and 10 mg/L were below the Limit of Detection or the Limit of Quantification. During the test the algae were exposed to a mean of 93 % of nominal. Therefore, all reported results refer to nominal concentrations. All validity criteria were met. The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test. The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test. The 72-hour ErC₅₀ value was calculated to be 73.0 mg test item/L and the 72-hour EyC₅₀ was calculated to be 17.5 mg test item/L. The 72-hour ErC₁₀ value was calculated to be 11 mg test item/L and the 72-hour EyC₁₀ was calculated to be 4.44 mg test item/L. The 72-hour NOErC was determined to be 3.2 mg test item/L and the associated 72-hour LOErC is 10 mg test item/L. The 72-hour NOEyC was determined to be lower than 3.2 mg test item/L and the associated 72-hour LOEyC is at least 3.2 mg test item/L.

Description of key information

The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test. The 72-hour ErC₅₀value was calculated to be 73.0 mg test item/L and the 72-hour EyC₅₀was calculated to be 17.5 mg test item/L. The 72-hour ErC₁₀value was calculated to be 11 mg test item/L and the 72-hour EyC₁₀was calculated to be 4.44 mg test item/L. The 72-hour NOErC was determined to be 3.2 mg test item/L and the associated 72-hour LOErC is 10 mg test item/L. The 72-hour NOEyC was determined to be lower than 3.2 mg test item/L and the associated 72-hour LOEyC is at least 3.2 mg test item/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

73 mg/L

EC10 or NOEC for freshwater algae:

11 mg/L

Additional information

Toxicity of the test item to Pseudokirchneriella subcapitata in an Algal Growth Inhibition Test determined according to OECD TG 201 and EU method C.3. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The study comprised of 6 treatment groups (5 dose rates of the test item, control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometric measurement. The samples of the test media sampled at the start and at the end of the test after 72 hours of exposure were analysed via TOC-method. At the start of the test 95 % of the nominal test concentrations were found (average of all test concentrations of nominal 32 to 320 mg/L). After 72 hours test duration, 90 % of the nominal value was determined (average of all test concentrations of nominal 32 to 320 mg/L). The test item concentrations of nominal 3.2 and 10 mg/L were below the Limit of Detection or the Limit of Quantification. During the test the algae were exposed to a mean of 93 % of nominal. Therefore, all reported results refer to nominal concentrations. All validity criteria were met. The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test. The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test. The 72-hour ErC₅₀ value was calculated to be 73.0 mg test item/L and the 72-hour EyC₅₀ was calculated to be 17.5 mg test item/L. The 72-hour ErC₁₀ value was calculated to be 11 mg test item/L and the 72-hour EyC₁₀ was calculated to be 4.44 mg test item/L. The 72-hour NOErC was determined to be 3.2 mg test item/L and the associated 72-hour LOErC is 10 mg test item/L. The 72-hour NOEyC was determined to be lower than 3.2 mg test item/L and the associated 72-hour LOEyC is at least 3.2 mg test item/L.

The reported effect concentrations refer to the commercial formulation containing 46.2% (w/w) of the REACH registration substance which reflects the test item used in the study.