Dimethyl[3-[(1-oxooctadecyl)amino]propyl][2-oxo-2-(tetradecyloxy)ethyl]ammonium chloride

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental start date was 12 Aug 2017, and the experimental completion date was 24 Nov 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006; Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl[3-[(1-oxooctadecyl)amino]propyl][2-oxo-2-(tetradecyloxy)ethyl]ammonium chloride

EC Number:

272-964-2

EC Name:

Dimethyl[3-[(1-oxooctadecyl)amino]propyl][2-oxo-2-(tetradecyloxy)ethyl]ammonium chloride

Cas Number:

68921-83-5

Molecular formula:

C39H79N2O3.Cl

IUPAC Name:

dimethyl(3-octadecanamidopropyl)[2-oxo-2-(tetradecyloxy)ethyl]azanium chloride

Constituent 2

Reference substance name:

Propane-1,2-diol

EC Number:

200-338-0

EC Name:

Propane-1,2-diol

Cas Number:

57-55-6

Molecular formula:

C3H8O2

IUPAC Name:

propylene glycol

Test material form:

semi-solid (amorphous): gel

Details on test material:

Identification Quaternium-70
Appearance Amber gel
Batch 0002000146
Composition UVCB
Test item storage at room temperature
Stable under storage conditions until 02 April 2018 (retest date)

Specific details on test material used for the study:

Identification: Quaternium-70
Appearance: Amber gel
Batch: 0002000146
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until 02 April 2018 (retest date)
Trade name: Ceraphyl 70

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

At the start of the test, samples were taken from the stock solutions in acetone for analytical confirmation of the prepared concentrations.

Single samples for possible analysis were taken from all test concentrations and the control.
Frequency at t=0 h, t=24 h and t=72 h
Volume 1.0 mL from the approximate centre of the test vessels.
Storage Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start and at the end of the test period.
The samples taken from the full test solutions and the nominal 0 mg/mL acetone stock solution were diluted in a 1:3 (v:v) ratio with acetonitrile and analyzed. If necessary, the samples were further diluted with 75/25 (v/v) acetonitrile/M2-medium to obtain concentrations within the calibration range. The other acetone stock samples were diluted by a factor of 100 with acetonitrile and thereafter by a factor of 2-2000 with 75/25 (v/v) acetonitrile/M2-medium.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of test solutions started with a stock solution of 100 mg Q-70/mL in acetone. No other treatment than vigorous shaking was needed to completely dissolve the test item in acetone. Stock solutions of lower concentrations were prepared by subsequent dilution of the highest stock concentration in acetone. Stock solutions of nominal 32 mg Q-70/mL and higher were clear and slightly yellow, while stock solutions of lower concentration were clear and colourless.
Subsequently, 100 µL of each stock were spiked into 1 litre of continuously stirred medium. Magnetic stirring was continued for 14-16 minutes to ensure complete dissolution of the test substance in test medium. Thereafter, the obtained solutions were allowed to settle before being used as test solutions (range-finding test: 65 minutes; full tests: 53-60 minutes). At the end of the preparation procedure, all test solutions were clear, colourless, and no Tyndall effect could be observed upon checking with a laser pen.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

Test organisms

Test organisms (species):

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

Details on test organisms:

Strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.
Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium M1: according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA).
Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was
measured immediately before use. Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 21 and 22°C.

pH:

8.1 ± 0.2

Nominal and measured concentrations:

Two full tests were performed following identical experimental protocols. At the end of the first full test, it could not be concluded with sufficient assurance that a sequential dilution series of stock solutions had been prepared, and thus it could not be considered that the test solutions had been prepared correctly. It was eventually decided to repeat the full test based on this finding.

Nominal: 0.010, 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L

Average measured (72h): the concentrations measured in the test solutions of either of the two tests perfomed did not reliably represent actual exposure concentrations of the test item due to the limited quality and repeatability of the analytical measurements. The fact that also in the repeated test, the analytical measurements were instable and not reliable, combined with the complexity of the UVCB and its limited solubility and stability in water, made it highly unlikely that analytical support for determination of actual exposure concentrations of this test item could be further improved. Analysis of the stock solutions proved sufficiently that the stock solutions had been prepared as a sequential dilution series.Hence, it was decided to relate the effects on algal growth to nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessels: 100 mL, all-glass, containing 50 mL of test solution
- Medium:       M2
- Cell density:  An initial cell density of 1 x 104 cells/mL.
- Illumination:   Continuously using-lamps with a light intensity within the range of 77 to 79 µE.m-2.s-1.
- Incubation:    Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- The pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 units).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval ranging from 0.23 to 0.86 mg Q-70/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.01 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

First full test:
S statistically significant difference was found between the blank control and the solvent control. Hence, the test concentrations were statistically compared to the solvent control group. The PG-control was statistically not different from the solvent control.
Inhibition of growth rates generally increased with increasing concentration. Statistically significant inhibition of growth rate was found at nominal concentrations of 0.032 mg Q-70/L and above.

Second full (Definitive) test:
No significant differences were recorded between the values for growth rate or yield in the solvent-control versus the blank control. Significant inhibition of growth rate occurred in the PG-control when compared to the pooled blank and solvent-control. Inhibition of growth rate and yield in the PG-control was statistically significant when compared to the pooled control group. However, the effect on growth rate was considered to be biologically not relevant (<10%).
IInhibition of growth rates increased generally with increasing concentration. Statistically significant inhibition of growth rate was found at nominal concentrations of 0.010 mg Q-70/L and above. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to nominally 0.010 mg Q-70/L when compared to the blank-control.

The statistical analysis of the biological results obtained from both full tests showed that the lowest concentration of nominally 0.010 mg Q-70/L did not significantly affect growth rate in the first test (4.4% reduction, not statistically significant at the 5% level) and reduced growth rate with 10% in the second test (statistically significant at the 5% level but not at the 1% level). Considering the overall comparability of the observed effects on algal growth inhibition in both full tests, it is justified to set the NOEC at the lowest tested nominal concentration of 0.010 mg Q-70/L.

Results with reference substance (positive control):

The EC50 for growth rate inhibition (72h-ERC50) was 1.1 mg/L with a 95% confidence interval ranging from 1.1 to 1.1 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.36 mg/L with a 95% confidence interval ranging from 0.35 to 0.36 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested was below this range. Nevertheless, this was accepted as the EC50 for growth rate was within historical range.

Any other information on results incl. tables

Growth rate and percentage inhibition during the definitive test:

  

Quaternium-70 Nominal conc. (mg Q-70/L)	Mean	Std. Dev.	n	%Inhibition
Pooled control	1.682	0.0269	12
PG-control	1.519	0.0509	6	9.7*^#
0.010	1.507	0.0721	3	10*
0.032	1.030	0.1235	3	39*
0.10	0.595	0.0702	3	65*
0.32	1.031	0.1657	3	39*
1.0	0.462	0.0801	3	73*
3.2	0.250	0.0471	3	85*
10	0.415	0.0865	3	75*

* effect was statistically significant;^#effect biologically not relevant (<10%)

Effect Parameters based on the definitive test:

	Parameter (mg Q-70/L)	NOEC	EC10	EC20	EC50
Growth rate	Value	0.010	n.d.	0.011	0.43
	lower 95%-cl		n.d.	0.003	0.23
	upper 95%-cl		n.d.	0.025	0.86

cl: confidence limit; n.d.: not determined;

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, Quaternium-70 reduced growth rate of this fresh water algae species significantly at nominally 0.032 mg Q-70/L and higher.
The EC50 for growth rate inhibition (72h-ErC50) was 0.43 mg Q-70/L with a 95% confidence interval ranging from 0.23 to 0.86 mg Q-70/L.
The 72h-NOEC for growth rate inhibition was 0.010 mg Q-70/L.

Executive summary:

The objective of the study was to evaluate Quaternium-70 for its ability to generate toxic effects in Pseudokirchneriella subcapitataduring an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀and EC₅₀for both inhibition of growth rate.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of Quaternium-70 tested was an amber gel and contained approximately 40% of propylene glycol. A factor of 1.67 was applied to correct for the purity/composition of the test item. Concentration/doses are expressed as mg Quaternium-70 per litre (mg Q-70/L).

Two full tests were performed according to identical procedures based on the results of a preceding combined limit/range-finding and an additional range-finding test. In both full tests, three replicates per group were exposed to 0.010, 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg Q-70/L. Control groups, including a blank, a solvent and a propylene glycol control, each consisted of six exponentially growing algal cultures.

Preparation of test solutions started with a stock solution of 100 mg Q-70/mL in acetone. Stock solutions of lower concentrations were prepared by subsequent dilution of the highest stock concentration in acetone.To obtain the final test concentrations, 100 µL of each stock were spiked into 1 litre of continuously stirred medium.Magnetic stirring was continued for 14-16 minutes to ensure complete dissolution of the test item in test medium. Thereafter, the obtained solutions were allowed to settle before being used as test solutions.At the end of the preparation procedure, all test solutions were clear, colourless, and no Tyndall effect could be observed upon checking with a laser pen.

The initial algal cell density was 10⁴cells/mL in all test vessels.The total exposure period was 72 hours. Samples for analytical confirmation of prepared concentrations were taken from the stock solutions in acetone at the start of the test and from the test solutions at the start, and after 24 and 72 hours of exposure.

Samples taken from all stock solutions and test concentrations were analysed employing two out of several peaks observed in the chromatograms of the test item, which was defined as a UVCB.In the stock solutions, the measured concentrations based on either of the two peaks showed a comparable profile of increasing well above 100% of the nominal concentration at lower concentration levels. The concentrations ranged from 98-179% of nominal according to the peak atm/z485.5 and, if quantifiable, from 107-153% of nominal based on the peak atm/z 623.6.

In the final test solutions, most concentrations measured according to either peak were below the limit of quantification throughout the test. Those concentrations that could be quantified at the start of exposure decreased below the applicable limit of detection or quantification at later sampling time points.

Due to the encountered analytical limitations, the effect parameters were based on nominal concentrations as being the minimum applied loading rates. Theanalysis of the stock solutions had sufficiently proven that the stock solutions had been prepared as a sequential dilution series.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, Quaternium-70 reduced the growth rate of this fresh water algae species significantly at nominally 0.032 mg Q-70/L and higher.

The EC₅₀for growth rate inhibition (72h-E_RC₅₀) was 0.43 mg Q-70/L with a 95% confidence interval ranging from 0.23 to 0.86 mg Q-70/L. The 72h-NOEC for growth rate inhibition was 0.010 mg Q-70/L.