Dodecyl(2-hydroxy-3-sulphonatopropyl)dimethylammonium

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 June 17th 2014 to July 25th 2014

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 2011-07-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Betadet S-20, termed 'AM20HD' in the test report.
Lot: 1671
Purity: 30.5% principal component (1-dodecanaminium, N-(2-hydroxy-3-sulfopropyl)N,N-dimethyl, inner salt)

Other constituents:
sodium chloride: 6.6%
sodium 1,2-dihydroxy-3-propanesulfonate: 2.8%
disodium 2-hydroxypropane-1,3-disulfonate 1.7%
N-(2,3-dihydroxypropyl)-N,N-dimethyl-N-dodecylammonium chloride: 1.4%
sodium epichlorohyrrinsulfonate: 0.8%
water: 56.1%

Analytical monitoring:

yes

Details on sampling:

Frequency of measurement: At the beginning and the end of exposure.

Samples (10 to 12 mL) were taken from the vessels used to prepare the test media at study initiation (0 h) and at termination from the replicate vessels of each treatment (72 h). Equal volumes were taken from each incubation vessel at the end of the test, centrifuged at 3000 rpm for 10 minutes to remove the algae, and mixed together according to treatment.

Vehicle:

no

Details on test solutions:

-Culture medium
OECD medium (OECD TG 201; March 23, 2006) prepared with purified water was used for preculture and tests.

-Preparation of the test solutions
A requisite amount of the test article was weighed and mixed/stirred with the culture medium to prepare a 100 mg/L stock solution (as a concentration of the main constituent). A requisite amount of the stock solution and the culture medium were mixed/stirred in a vessel for preparation to prepare a test solution. Then, the test solution was aliquoted into each test vessel.

Test organisms (species):

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

Details on test organisms:

Reasons for selecting the species: This species was recommended in the test guideline.
Supplier: American Type Culture Collection
Cell line number: ATCC 22662
Date of receipt: June 30, 1995
Maintenance after receipt: Subcultured aseptically at the test facility

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not relevant.

Hardness:

Not applicable

Test temperature:

From 22.5 to 22.8°C

pH:

From 7.7 to 8.0

Dissolved oxygen:

Not applicable

Salinity:

Not applicable (freshwater test)

Conductivity:

Not applicable

Nominal and measured concentrations:

-Nominal concentrations: 0 (control), 0.616, 1.67, 4.49, 12.1 and 32.8 mg Betadet S-20 ('AM20HD')/L, corresponding to 0, 0.188, 0.508, 1.37, 3.70 and 10.0 mg main component/L.
-Mean measured concentrations:

Details on test conditions:

-Test conditions
Exposure procedure: Orbital shaking culture (approximately 100 rpm)
Exposure time: 72 hours
Control culture: Medium without test article
Number of replicates: 6 replicates/control culture
3 replicates/test culture
Amount of test solutions: 600 mL/control culture (100 mL/vessel)
300 mL/test culture (100 mL/vessel)
Number of cells at the beginning of exposure: The number of cells of algae precultured for 3 days from June 14, 2014 to June 17, 2014 was counted, and the cells were inoculated in the test solution so that the cell concentration might be 0.75×104 cells/mL.
Test procedures: The test was conducted under sterile conditions.
Lighting: Set value, 90 μmol/m2/s (within ± 20% of the set value, fluctuations within the mean±15%)
Continuous illumination provided with fluorescent lamps in the spectral range of 400 to 700 nm.

- Observations and measurement
a) Algal growth
Measurement items: Biomass (cell concentrations)
Frequency of measurement: Measured every 24 hours after the beginning of exposure (The background of the test solution was determined, and blank correction was done)
Observations of cells: One test vessel per test culture at the end of exposure
Measuring instrument: Coulter Counter model Z1 (Beckman coulter)
Microscope BX41 (Olympus)

b) State of the test solution
Observed at the beginning and the end of exposure.

c) Water quality of the test solution and exposure environment
pH: Measured a portion of the test solution separated from the vessels for preparation (at the beginning of exposure)
Measured one test vessel per test culture (at the end of exposure)
Temperature in the culturing apparatus: Measured once a day during the exposure period
Light intensity: Measured in the culturing apparatus once a day during the exposure period
Measuring instrument: pH meter (model HM-21P, DKK-TOA Co.)
Glass stick thermometer
Light quantum meter (LI-250A) (LI-COR)

-Effectiveness of the test
a) The cell concentration in the control culture should have increased by a factor of at least 16 within 72 hours.
b) The mean coefficient of variation for growth rates on each day in the control culture must not exceed 35% throughout the exposure period.
c) The coefficient of variation for growth rates in the replicate control cultures must not exceed 7%.

-Calculation of the results
The nominal concentrations were used to calculate results since the concentration of the test substance in the test solution determined during the exposure period was within ± 20% of the nominal concentration.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.58 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% CL 4.34 to 4.83 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.508 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Betadet S-20

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 14.2 to 15.2 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Betadet S-20

Basis for effect:

growth rate

Details on results:

-Results of observations and measurements of the test solutions
a) State of the test solutions
The test solution in each test culture was clear and colorless at the beginning of exposure. At the end of exposure, the test solution was clear and colorless in the test culture containing a concentration of 10.0 mg/L, subtle green due to cell growth in the test culture containing a concentration of 3.70 mg/L, pale green in the test culture containing a concentration of 1.37 mg/L, green in the test culture containing a concentration of 0.508 mg/L and slightly dark green in the test culture containing a concentration of 0.188 mg/L. The test solution in the control culture was clear and colorless at the beginning of exposure and green due to cell growth at the end of exposure.

b) Water quality of the test solution and exposure environment
The light intensity in the culturing apparatus were within a range from 92 to 94 μmol/m2/s, respectively.

c) Concentrations of the main constituent in the test solutions
The concentrations of the main constituent in the test solutions were within a range from 0.184 to 9.72 mg/L at the beginning of exposure and from 0.175 to 10.1 mg/L at the end of exposure. Those concentrations were 96.7 to 101% and 93.2 to 101% of the nominal concentrations, respectively and were within ± 20% of the nominal concentration.

-Growth curve, the result of observations of cells and NOEC in each test culture
Cell growth was markedly inhibited throughout the exposure period in the test culture containing a concentration of 10.0 mg/L. Cell growth was inhibited but gradually increased in the test culture containing a concentration of 3.70 mg/L. Cell growth inhibition was observed but logarithmic growth was observed in the test culture containing a concentration of 1.37 mg/L. Cell growth similar to that in the control culture was shown in the test culture containing a concentration of 0.508 and 0.188 mg/L.

These results of observations of cells were based on comparison to the control culture. Many swollen cells and collapsed cells were observed in the test culture containing a concentration of 10.0 mg/L. Many swollen cells were observed in the test culture containing a concentration of 3.70 mg/L and a few swollen cells were observed in the test culture containing a concentration of 1.37 mg/L. The cells in the other test cultures were observed similarly to those in the control culture. No abnormalities were observed in the control culture.
In the significance test for growth rates, a statistically significant difference was observed in the test cultures containing a concentration of 3.70 and 1.37 mg/L, respectively. Based on the results of the significance test and observations of cells described above, NOEC based on the growth rate was 0.508 mg/L.

-Effectiveness of the test
a) Growth in the control culture
Logarithmic growth of the test algae was observed in the control culture until the end of exposure. At the end of exposure, the cell concentration increased by a factor of at least 132 compared to the initial biomass concentration and met the criteria for effectiveness (increases by a factor of at least 16).
b) Daily growth rate in the control culture
The mean coefficient of variance for daily growth rates in the control culture was 11% and met the criteria for effectiveness (should not exceed 35%).

c) Growth rate in the replicate control cultures
The coefficient of variance for growth rates in the replicate control cultures was 0.37% and met the criteria for effectiveness (should not exceed 7%).

-Environmental factors that were considered to affect the reliability of the study results
There was no relevant factor.

Results with reference substance (positive control):

Confirmation of reproducibility of the test system:
An algal growth inhibition test of the reference substance is regularly conducted. The latest data are shown as follows.
Reference substance: Potassium dichromate
(Wako Pure Chemical Industries Ltd., Special grade chemicals, Lot No. RCK7838)
Study period: May 19, 2014 to May 22, 2014
ErC50 (0-3d): 1.1 mg/L
This value was within the reference range (mean ± 2 × standard deviation) in our institution’s background data [mean ± standard deviation: 0.96 ± 0.17 mg/L (n=24)].

Results of the analysis of test media at the start and end of the incubation are presented below.

Table 1: Measured concentrations of the main component in a study of the effects of Betadet S‑20 ('AM20HD') on the growth of P. subcapitata

Nominal concentration (mg/L)		Measured concentration of main component (mg/L)
Betadet S‑20	main component	0 h	72 h	geometric mean [% of nominal]
Control	-	n.d.	n.d.	-	[-]
0.616	0.188	0.184	0.175	0.180	[95.6]
1.67	0.508	0.515	0.480	0.498	[97.9]
4.49	1.37	1.32	1.34	1.33	[97.4]
12.1	3.70	3.63	3.72	3.68	[99.3]
32.8	10.0	9.72	10.1	9.90	[99.0]
n.d.:       <0.0250 mg/L

 

Algal cell densities recorded at intervals during the incubation, calculated specific growth rates and corresponding levels of inhibition relative to the untreated control are prsented below.

Table 2: Measured algal cell concentrations and calculated growth rate inhibition in a study of the effects of Betadet S‑20 ('AM20HD') on the growth of P. subcapitata

Nominal concentration (mg/L)		Mean algal cell density (×10^4cells/mL)				Mean specific growth rate (μ) 0 – 72 h	Inhibition compared to control (%)
Betadet S‑20	main component	0 h	24 h	48 h	72 h
control	-	0.75	3.2	20	100	1.64	n.a.
0.616	0.188	0.75	3.3	21	110	1.66	‑1.4
1.67	0.508	0.75	3.1	19	100	1.64	‑0.0099
4.49	1.37	0.75	2.7	16	76	1.54**	6.1
12.1	3.70	0.75	1.7	5.5	20	1.07**	34
32.8	10.0	0.75	1.6	5.1	19	‑0.115†	107
**           Significantly different from the control (p<0.01), Bartlett’s test, one-way ANOVA and Dunnett’s multiple comparison test. †            Treatment excluded from statistical analysis because it exceeded the ErC50.

 

Validity criteria fulfilled:

yes

Conclusions:

The 72 h ErC50 of Betadet S-20 (termed 'AM20HD' in the report) that caused a 50% reduction in growth rate of Psudokirchneriella subcapitata compared to the untreated control group was 4.58 mg/L (95% CL 4.34 to 4.83 mg/L) based on analytically confirmed nominal concentrations of the principal constituent. The corresponding NOErC was 0.508 mg principal constituent/L.

Executive summary:

The effects of Betadet S-20 (tested as AM20HD) on the growth rate of the unicellular green alga Pseudokirchneriella subcapitata were determined in a static study carried out according to OECD test guideline 201 (2011). Algal cells were exposed to nominal Betadet S-20 concentrations of 0 (control), 0.616, 1.67, 4.49, 12.1 and 32.8 mg/L, corresponding to 0, 0.188, 0.508, 1.37, 3.70 and 10.0 mg principal constituent/L. Analysis confirmed that the nominal concentrations of the principal constituent were achieved and were adequately maintained during the incubation. The biological results of the study were therefore related to nominal concentrations expressed in terms of the principal constituent.

The 72h ErC50 of Betadet S-20 was calculated to be 4.58 mg/L (95% CL 4.34 to 4.83 mg/L) based on nominal concentrations of the principal constituent. The corresponding 72 h NOErC was 0.508 mg principal constituent/L.

Description of key information

The 72 h EC50 (ErC50) was calculated to be 4.58 mg/L (95% confidence limit: 4.34 to 4.83 mg/L) and the corresponding NOEC (growth speed: 0-3d) was 0.508 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.58 mg/L

EC10 or NOEC for marine water algae:

0.508 mg/L