Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1)

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:

17 December 2017 to 11 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006, corrected 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations, including control
- Sampling method: Analytical samples were taken in the main test at 0 hours (initial value) from fresh test solutions and after 72 hours from aged test solutions from all test item concentrations and control. For each sampling timepoint, a retained sample was also taken.
- Sample storage conditions before analysis: All samples were stored deep frozen until they were transferred to the analytical laboratory.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fraction (WAF) - The test item was applied as water accommodated fraction (WAF). Stock solutions (S1, S2) were prepared by directly weighing the necessary amount of test item in test medium. These stock solutions were stirred in the dark at room temperature for 48 h (based on OECD Series on Testing and Assessment No. 23). The solutions were clear and transparent. After the settling of one hour Tyndall’s effect could be observed for S1 and S2. The stock solutions were centrifugated for one hour at 5000 rpm. Afterwards only S1 showed a slight Tyndall`s effect. Lower test solutions were prepared by dilution of appropriate solutions with test medium.
- Eluate: AAP-Medium
- Differential loading: 10.0, 3.13, 0.977, 0.305, 0.0954 mg/L
- Controls: Test medium only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Not present in test solutions used for study (aqueous phase of WAF siphoned off)

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Not applicable
- Strain: SAG 61.81
- Source (laboratory, culture collection): laboratory culture
- Age of inoculum (at test initiation): 3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth.
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are ordered regularly from our commercial supplier.

ACCLIMATION
- Acclimation period: Not applicable (laboratory culture)
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: None reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Stabdard guideline exposure duration

Test temperature:

22.2 – 23.6 °C

pH:

7.38 – 8.13

Nominal and measured concentrations:

Nominal Concentrations: 10.0, 3.13, 0.977, 0.305, 0.0954 mg/L and control

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): closed (aluminium caps)
- Material, size, headspace, fill volume: glass, 100 mL, 50 mL
- Aeration: CO2 supply by continuous agitation: Test vessels were placed in an incubator on a pivoted bogie which turns around and induces shaking by regular sudden stops.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 0.5 E +04
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes AAP-Medium (according to Annex 3 of OECD 201)
- Detailed composition if non-standard medium was used: Not applicable

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No
- Intervals of water quality measurement: Measurements of pH-value were performed at t = 0 h and t = 72 h, the temperature was measured continuously and recorded at hours 0, 24, 48 and 72 h. The light intensity of all positions of the incubator is measured once a year and was confirmed for one representative position at test start.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: No
- Photoperiod: 24 h light
- Light intensity and quality: Continuously, 88.8 µEm-2s-1 (mean)
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED
Growth determination was performed by fluorescence measurement.
The fluorescence measurements were performed with a fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.11.1.0).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL: 2.34 - 2.99

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1.03 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL: 0.737 - 1.28

Details on results:

After 72 h at termination of the test a dose response relation was observed for the inhibition of growth rate and yield from nominal test item concentrations of 0.305 to 10.0 mg/L. The inhibition of growth peaked in 139.9 % and the inhibition of yield peaked in 101.2 % at a nominal test item concentration of 10.0 mg/L.
The morphology of the algae cells was observed microscopically at test end. The cells were considered normal for the control and up to and including a nominal test item concentration of 0.305 mg/L. Spherical cells were noted at 0.977 and 3.13 mg/L. Additionally for test item concentration 3.13 mg/L less cells were found. No cells were observed at 10.0 mg/L nominal test item concentration.
The growth conditions (pH and temperature) during the test were within the range specified by OECD 201.
The mean light intensity of all positions of the incubator was 88.8 µEm-2s-1 with mean values of each platform in a range from 82.6 to 93.6 µEm-2s-1 (-7.0 % to +5.4 % of mean) which was within ± 15 % of variation as specified by OECD 201.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.971 mg/L

Reported statistics and error estimates:

The statistical evaluation for the 72 hours period was performed for growth rate and yield using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOELR and LOELR were determined by using a multiple comparison method (Jonckheere Terpstra test, two tailed, for growth rate, Bonferroni-Holms corrected Welch test, two tailed, for yield). The EC10, 20, 50-values for growth rate and yield were determined by probit analysis following the Gompertz and normal distribution, respectively. Due to statistical reasons inhibition-values above 100 % were set to 100 and values below 0 % were set to zero. Only concentrations within a clear concentration response relation were used for calculations.

The measured initial loading rates of amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) ranged from 94 % to 103 % of nominal with a mean initial loading rate of 99 % of nominal. In the aged samples the measured loading rates were between 92 % and 104 % of nominal with a mean measured loading rate of 98 % of nominal. Toxicological endpoints were evaluated using the nominal loading rates of the test item.

Table 1. Main Test: % Inhibition of Growth Rate

	% Inhibition of Growth Rate
Loading Rate (mg/L)	0 h - 24 h	0 h - 48 h	0 h - 72 h
Control	-31.3 14.1 -41.8 24.4 5.6 28.9	-8.5 1.4 -13.5 7.7 4.3 8.5	-5.3 2.0 -6.3 -2.4 4.0 7.9
Mean	0.0	0.0	0.0
0.0954	2.4 30.6 -17.8	-0.5 18.0 -4.5	3.9 8.2 0.8
Mean	5.1	4.3	4.3
0.305	-36.0 -26.4 -32.9	-20.5 -10.8 -19.4	-8.9 -2.9 -7.5
Mean	-31.8	-16.9	-6.4
0.977	-31.5 23.4 1.7	-6.5 0.6 3.2	8.2 12.4 11.7
Mean	-2.1	-0.9	10.8
3.13	85.0 -22.9 73.1	82.4 29.0 37.2	84.5 48.7 51.1
Mean	45.1	49.5	61.5
10.0	184.6 125.4 125.4	101.2 172.9 157.1	143.8 135.9  -
Mean	145.1	143.8	139.9

Table 2. Determined loading rate of amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1)

Test Item Nominal (mg/L)	Sampling Time Point	Concentration (mg/L)	% of nominal
Control	0 h fresh 72 h aged	< LOD < LOD	- -
0.0954	0 h fresh 72 h aged	0.0981 0.0946	103 99
0.305	0 h fresh 72 h aged	0.304 0.294	100 06
0.977	0 h fresh 72 h aged	0.920 0.897	94 92
3.13	0 h fresh 72 h aged	3.22 3.10	103 99
10.0	0 h fresh 72 h aged	9.62 10.4	96 104

Validity criteria fulfilled:

yes

Conclusions:

The EL10-value for growth rate (ErL10) was determined to be 1.03 mg/L (nominal).
The EL50-value for growth rate (ErL50) was determined to be 2.64 mg/L (nominal).

Executive summary:

The study was conducted to determine the effects of the test item on the growth of the single cell green alga Pseudokirchneriella subcapitata, according to OECD 201. Initial target cell densities of 0.5 × 10⁴ cells/mL were employed for the individual replicates. The increase of cell numbers was assessed over a test period of 72 hours. Inhibition of growth was assessed by the determination of NOELR/LOELR and EL10, 20, 50 for growth rate and yield after 72 hours. Following a static GLP range-finding test with nominal test item loading rates of 100, 10.0, 1.00 mg/L and control a static main test with nominal test item loading rates of 10.0, 3.13, 0.977, 0.305, 0.0954 mg/L and control was performed. Six replicates were employed for the control and three for each test item loading rate. The test was performed in 100 mL Erlenmeyer flasks each containing ~ 50 mL test solution. The pH was recorded at test start and test end. Temperature was measured continuously over the whole test period and recorded daily. Light intensity of the continuous illumination was measured at test start. Analytical samples were taken and analysed in the main test from control and all test item loading rates at 0 hours (initial value) from fresh test solutions and after 72 hours from aged test solutions. Where possible NOELR and LOELR were determined by using a multiple comparison method and EL10, 20, 50-values were determined by probit analysis. Biomass: Cell numbers, measured in the controls between 0 h and 72 hours, were found to increase by a factor of 72.86, which exceeds the threshold of 16. It corresponds to a growth rate of 1.42438 d-1. Coefficient of Variation (section by section): The mean coefficient of variation for the section-by-section specific growth rates (hours 0 - 24, 24 - 48 and 48 - 72) in the control cultures was 30 % and did not exceed 35 %. Coefficient of Variation (average growth rate): The coefficient of variation of average growth rate in replicate control cultures was 5.6 % and did not exceed 7 % for the whole test period. The pH-value of the control ranged from 7.38 to 8.19 during the test period, the temperature was measured to be 22.2 – 23.6 °C during the test and the mean light intensity was 88.8 µEm-2s-1 at cell culture level. The measured initial loading rates of amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) ranged from 94 % to 103 % of nominal with a mean initial loading rate of 99 % of nominal. In the aged samples the measured loading rates were between 92 % and 104 % of nominal with a mean measured loading rate of 98 % of nominal. Toxicological endpoints were evaluated using the nominal loading rates of the test item. The EL10-value for growth rate (ErL10) was determined to be 1.03 mg/L (nominal). The EL10-value for yield (EyL10) was 0.572 mg/L (nominal). The EL50-value for growth rate (ErL50) was determined to be 2.64 mg/L (nominal). The EL50-value for yield (EyL50) was 1.23 mg/L (nominal).

Description of key information

72 h ErL50 = 2.64 mg/L, 72 h ErL10 = 1.03 mg/L; OECD 201 (Pseudokirchneriella subcapitata); Schuster, 2018

Key value for chemical safety assessment

EC50 for freshwater algae:

2.64 mg/L

EC10 or NOEC for freshwater algae:

1.03 mg/L

Additional information

OECD 201 (2018): A study was conducted to determine the effects of the test item on the growth of the single cell green alga Pseudokirchneriella subcapitata, according to OECD 201. Initial target cell densities of 0.5 × 10⁴ cells/mL were employed for the individual replicates. The increase of cell numbers was assessed over a test period of 72 hours. Inhibition of growth was assessed by the determination of NOELR/LOELR and EL10, 20, 50 for growth rate and yield after 72 hours. Following a static GLP range-finding test with nominal test item loading rates of 100, 10.0, 1.00 mg/L and control a static main test with nominal test item loading rates of 10.0, 3.13, 0.977, 0.305, 0.0954 mg/L and control was performed. Six replicates were employed for the control and three for each test item loading rate. The test was performed in 100 mL Erlenmeyer flasks each containing ~ 50 mL test solution. The pH was recorded at test start and test end. Temperature was measured continuously over the whole test period and recorded daily. Light intensity of the continuous illumination was measured at test start. Analytical samples were taken and analysed in the main test from control and all test item loading rates at 0 hours (initial value) from fresh test solutions and after 72 hours from aged test solutions. Where possible NOELR and LOELR were determined by using a multiple comparison method and EL10, 20, 50-values were determined by probit analysis. Biomass: Cell numbers, measured in the controls between 0 h and 72 hours, were found to increase by a factor of 72.86, which exceeds the threshold of 16. It corresponds to a growth rate of 1.42438 d-1. Coefficient of Variation (section by section): The mean coefficient of variation for the section-by-section specific growth rates (hours 0 - 24, 24 - 48 and 48 - 72) in the control cultures was 30 % and did not exceed 35 %. Coefficient of Variation (average growth rate): The coefficient of variation of average growth rate in replicate control cultures was 5.6 % and did not exceed 7 % for the whole test period. The pH-value of the control ranged from 7.38 to 8.19 during the test period, the temperature was measured to be 22.2 – 23.6 °C during the test and the mean light intensity was 88.8 µEm-2s-1 at cell culture level. The measured initial loading rates of amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) ranged from 94 % to 103 % of nominal with a mean initial loading rate of 99 % of nominal. In the aged samples the measured loading rates were between 92 % and 104 % of nominal with a mean measured loading rate of 98 % of nominal. Toxicological endpoints were evaluated using the nominal loading rates of the test item.  The EL10-value for growth rate (ErL10) was determined to be 1.03 mg/L (nominal). The EL10-value for yield (EyL10) was 0.572 mg/L (nominal).  The EL50-value for growth rate (ErL50) was determined to be 2.64 mg/L (nominal). The EL50-value for yield (EyL50) was 1.23 mg/L (nominal).