4,4-Dimethyl-2-undecyl-2-oxazoline

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:

12th June 2017 - 22nd December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP, Guidelines : OECD 201

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

This deviation is considered to have no impact on the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,4-dimethyl-2-undecyl-4,5-dihydro-1,3-oxazole

EC Number:

686-853-5

Cas Number:

46921-17-9

Molecular formula:

C16H31NO

IUPAC Name:

4,4-dimethyl-2-undecyl-4,5-dihydro-1,3-oxazole

impurity 1

Reference substance name:

Unknown impurities

IUPAC Name:

Unknown impurities

impurity 2

Reference substance name:

IUPAC name not applicable

Molecular formula:

C16H33NO2

IUPAC Name:

IUPAC name not applicable

impurity 3

Reference substance name:

IUPAC name not applicable

Molecular formula:

C28H55NO3

IUPAC Name:

IUPAC name not applicable

Test material form:

liquid

Specific details on test material used for the study:

Purity : 88,0% / Storage conditions : stored in closed cabinet away from light at room temperature (15-25°C).

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Five concentrations up to the water solubility limit were prepared and divided in four replicates. Three replicates were inoculated with algae strain and incubated in a phytotronic
chamber during 72h. the fourth replicate were used as colour control. After 24, 48 and 72 hours, optical density were measured (683 nm) to determine cells concentration in each replicate. An aliquot was taken in each concentration preparation at t0, t24h, t48h and t72 for Lauryl Oxazoline quantification.

Test solutions

Vehicle:

no

Details on test solutions:

The test item stock solutions (water solubility level) were prepared by dissolution of about 4 x 5 mg of Lauryl Oxazoline in 4 x 1000 mL of the test media. The solutions were prepared in 2 L glass bottle and shacked during about 24h. Then these preparations were decanted at about 5 minutes in separating funnel, filtered through 0.2 μm filter and mixed. This solution
was used as water solubility level and diluted in media to prepare several dilutions. The pH of this solutions was 7.9. Test media solution pH was between 6 and 9 considered as appropriate for a normal life of test organisms.

Test organisms

Test organisms (species):

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

Details on test organisms:

Culture conditions :
Temperature: 23°C ± 2°C. Light: continuous, with plant growth specific spectra fluorescent bulb, between about 4440 and 8880 Lux. Freshwater (reconstituted from salts). The algae are maintained at exponential growth rate by adding aliquots of algal culture to fresh reconstituted water samples weekly.

Origin :
Culture at SGS Multilab Rouen, strain from SAG Gottingen collection (ID n° 61.81).

Study design

Test type:

static

Water media type:

freshwater

Remarks:

reconstituted from salts

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

The test solutions at T0, T24h, T48h and T72h were sampled (volume: 200mL), extracted and quantified immediately according to the validated method.

Post exposure observation period:

Not applicable

Test conditions

Test temperature:

23,7 to 24,9°C. The test was carried out in a phytotronic incubator at 23°C controlled at ± 2°C (temperature was recorded automatically by Evisense Temperature Center).

pH:

Test media solution pH was between 6 and 9 considered as appropriate for a normal life of test organisms. pH Variation in control solutions (pH units) : 0.4. Criteria : ≤ ± 1.5.

Nominal and measured concentrations:

Nominal concentrations:
The definitive test was performed with five concentrations up to water solubility limit (SL) and with a dilution factor of 2 (SL, SL/2, SL/4, SL/8, SL/16).
SL = 0,500 mg/L ;
SL/2 = 0,250 mg/L ;
SL/4 = 0,125 mg/L ;
SL/8 = 0,0625 mg/L ;
SL/16 = 0,03125 mg/L.

Measured concentrations (T0h):
SL = 0,5892 mg/L ;
SL/2 = 0,2863 mg/L ;
SL/4 = 0,1382 mg/L ;
SL/8 = 0,0709* mg/L ;
SL/16 = 0,0419 mg/L.

Measured concentrations (T24h):
SL = 0,3724 mg/L ;
SL/2 = 0,1706 mg/L ;
SL/4 = 0,1012 mg/L ;
SL/8 = 0,0401 mg/L ;
SL/16 = 0,0122 mg/L.

Measured concentrations (T48h):
SL = 0,2847 mg/L ;
SL/2 = 0,0420 mg/L ;
SL/4 = 0,0095 mg/L ;
SL/8 = SL/16 =
Measured concentrations (T72h):
SL = 0,0638 mg/L ;
SL/2 = SL/4 = SL/8 = SL/16 =
LOQ = 0.01 mg/L
*Extrapolated value, measured concentration were unvalidated.

Details on test conditions:

- No. of organisms per vessels : 5 000 to 10 000 cells per mL
- No. of vessels per concentration (replicate) : 5
No. of vessels per control : 7 (A control (7 replicates) were performed at the same time with test media only. One replicate was used for quantification purpose only (800 to 1000 mL)).
- Photoperiod : continuous light
- Source/preparation of dilution water : prepared as described in OECD 201 test guideline

Reference substance (positive control):

yes

Remarks:

potassium dichromate (99,99%)

Results and discussion

Effect concentrations

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

and EC10

Effect conc.:

ca. 0.046 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

(see tables below in the section : "Any other information on results incl. tables")

Reported statistics and error estimates:

(see tables below in the section : "Any other information on results incl. tables")

Any other information on results incl. tables

Growth rate and yield Inhibition per concentration :

Nominal concentrations (mg/L)	Growth rate (day-1)	Growth inhibition (%)	Yield (cells/mL)	Yield inhibition (%)
Media control	1.8899	0.0	1378604	0.0
SL	0.3364	82.2	8845	99.4
SL/2	1.0522	44.3	123750	91.0
SL/4	1.6076	14.9	598497	56.6
SL/8	1.7487	7.5	925395	32.9
SL/16	1.8346	2.9	1183246	14.2

Reported statistics and error estimates / NOEC and LOEC determination for growth inhibition (using Statgraphics(R)) :

Results of nominal distribution for growth rate:

Test item nominal concentration (mg/L)	Effectif	Average	Standard-deviation	Skewness	kurtosis	Normal distribution
Control	6	1.890	0.044	0.060	-1.044	yes
SL/16	3	1.835	0.078	1.193	-	yes
SL/8	3	1.749	0.097	0.680	-	yes
SL/4	3	1.608	0.067	0.713	-	yes
SL/2	3	1.052	0.192	0.996	-	yes
SL	3	0.336	0.062	-0.653	-	yes

Test of Levene for growth rate :

	Test	Probability	Target probability	Homoscedasticity
Levene test	0.7098	0.6253	>= 0.05	Yes

Data were checked for distribution compatible with ANOVA analysis.

Test of ANOVA for growth rate :

ANOVA TABLE	Square sum	Dof	Average square	F	Probability	Target probability	Homogeneous groups
Inter group	6.1232	5	1.2246	140.05	0	>= 0.05	No
Intra group	0.1312	15	0.0087	-	-	-	-
Total	6.2544	20	-	-	-	-	-

As ANOVA analysis do not lead to conclude to homogeneous groups, an effects was detected in relation to growth rate. A Tukey HSD pairwise test was made to find homogeneous groups.

Test of Tukey HSD pairwise for growth rate :

Contrast	Difference	+/- limits	Homogeneous group
Control - SL/16	0.0553	0.2153	Yes
Control - SL/8	0.1411	0.2153	Yes
Control - SL/4	0.2823	0.2153	No
Control - SL/2	0.8376	0.2153	No
Control - SL	1.5535	0.2153	No
SL/16 - SL/8	0.0858	0.2487	Yes
SL/16 - SL/4	0.2270	0.2487	Yes
SL/16 - SL/2	0.7823	0.2487	No
SL/16 - SL	1.4982	0.2487	No
SL/8 - SL/4	0.1412	0.2487	Yes
SL/8 - SL/2	0.6965	0.2487	No
SL/8 - SL	1.4124	0.2487	No
SL/4 - SL/2	0.5553	0.2487	No
SL/4 - SL	1.2712	0.2487	No
SL/2 - SL	0.7159	0.2487	No

Tukey HSD pairwise analysis leads to conclude that there was probably not difference between control condition and SL/8 (NOEC = 0.0278 mg/L) but detected a significant difference between control condition and SL/4 (LOEC = 0.0622 mg/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Ecotoxicity test of Lauryl Oxazoline on Pseudokirchneriella subcapitata fulfils the requirements of OECD test No. 201 to determine EC50y-72h = 0.046 mg/L [0.034 - 0.060] mg/L.

Executive summary:

Objective

The scope of this study was, in compliance with the principles of Good Laboratory Practices

(GLP), to determine the acute toxicity of Lauryl Oxazoline on Pseudokirchneriella subcapitata

(Algae) in a 72-hours test.

 

Method Summary

Five concentrations up to the water solubility limit were prepared and divided in four

replicates. Three replicates were inoculated with algae strain and incubated in a phytotronic

chamber during 72h. the fourth replicate were used as colour control. After 24, 48 and 72

hours, optical density were measured (683 nm) to determine cells concentration in each

replicate. An aliquot was taken in each concentration preparation at t0, t24h, t48h and t72 for

Lauryl Oxazoline quantification.

 

A control (6 replicates) was performed at the same time with test media only.

	Lauryl Oxazoline BPL16 -0047.001 Based on measured concentrations
ErC50 -72h [Confidence limits 95%]	0.146 mg/L [0.133 - 0.160] mg/L
ErC10 -7h2h  [Confidence limits 95%]	0.046 mg/L [0.037 - 0.057] mg/L
EyC50 -72h  [Confidence limits 95%]	0.046 mg/L [0.034 - 0.060] mg/L
EyC10 -72h  [Confidence limits 95%]	0.013 mg/L 0.006 - 0.023] mg/L
NOECy	0.0135 mg/L
LOECy	0.0278 mg/L

ECX-72h: Effective Concentration corresponding to X% of mobility inhibition in the 72 -hours test

r : growth rate

y : growth yield

NOEC : No observed Effect concentration

LOEC : Lowest Observed Effect Concentration

Conclusion

Ecotoxicity test of Lauryl Oxazoline on Pseudokirchneriella subcapitata fulfils the

requirements of OECD test No. 201 to determine EC50y-72h = 0.046 mg/L [0.034 - 0.060]

mg/L.