Reaction mass of methyl N-octadecylterephthalamate and 37437-26-6

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:

2016-03-14 to 2016-03-18

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of methyl N-octadecylterephthalamate and 37437-26-6

EC Number:

943-498-3

Molecular formula:

C27 H45 N O3 C44 H80 N2 O2

IUPAC Name:

Reaction mass of methyl N-octadecylterephthalamate and 37437-26-6

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

Water accommodated fraction
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

Preparation of test solutions
For the single water accommodated fraction (WAF) 100 mg/L of the test item was weighed out and transferred into a brown glass flask with dilution water one day prior to application. The WAF was shaken with 20 rpm for 24 hours at room temperature. After a separation phase of 1 hour, the WAF was taken from the homogeneous liquid phase from the middle of the water body.

Test loading
Per definition of the WAF all terms related to concentration level are given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations. The water accommodated fraction (WAF) was tested in a limit test.

One limit loading of 100 mg/L was tested. The limit loading is based on the results of a preliminary range finding test (non- GLP).

Control
Six replicates (without test item) were exposed under the same conditions as the test item replicates.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 21.0 max.: 22.5 mean value: 21.8

pH:

Nominal Test Item Loading pH-value
[mg/L] Start; 0 hours End; 72 hours
100 7.90 8.38
Control 8.06 9.01

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

No analytical monitoring

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 5 x 10^3 - 10^4 cells/mL, Actual: mean 6305 cells/mL
- Control end cells density: Mean 2126101 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6




GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m^-2 x s^-1, mean value: 72.7 µE x m^-2 x s^-1,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA-ALDRICH, batch number MKBQ9179V, purity 100 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2015-10-13 to 2015-10-16. The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item based on nominal concentrations mg/L, (0-72 hours)

Growth Rate inhibition
ErC50: 0.564 mg/L
95 % confidence interval: 0.546 - 0.582 mg/L
Current Study Valid Range (average ± 3 x SD): 0.797 ± 0.428 mg/L

Yield inhibition
EyC50: 0.322 mg/L
95 % confidence interval: 0.239 - 0.342 mg/L
Current Study Valid Range (average ± 3 x SD): 0.415 ± 0.275 mg/L

Reported statistics and error estimates:

EL-values and statistical analyses
EL-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).

NOEL, LOEL and statistical analyses
The NOEL and LOEL were determined by calculation of statistically significant differences of growth rates and yield. A t-test was used for NOEL/LOEL calculations. When running a t-test, a Normality test and an Equal Variance test were done first. P-values for both normality and equal variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the fully derived data. Consequently, if calculatedmanually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.

Any other information on results incl. tables

Cell Densities

Nominal test item loading	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
100	1	6305	28806	270175	1453647
	2	6305	29813	293944	1670203
	3	6305	33131	331009	1491873
	4	6305	33043	298793	1523446
	5	6305	27687	350449	1688019
	6	6305	29987	356233	1665411
	Mean	6305	30411	316767	1582100
Control	1	6305	34819	421217	2115605
	2	6305	31228	361533	2098465
	3	6305	34261	408915	2347665
	4	6305	28894	370779	1987904
	5	6305	35921	435255	2190534
	6	6305	36274	407302	2016432
	Mean	6305	33566	400834	2126101

  

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

 

Nominal test item loading		Replicate		Growth rate			Inhibition of growth rate		Yield			Inhibition of yield
[mg/L]		No.		[d^-1]			[%]		[cells/mL]			[%]
100		1				1.81		7				1447342		32
		2				1.86		4				1663898		22
		3				1.82		6				1485568		30
		4				1.83		6				1517141		28
		5				1.86		4				1681714		21
		6				1.86		4				1659106		22
		Mean		(+)*		1.84		5		(+)		1575795		26
Control		1				1.94						2109300
		2				1.94						2092160
		3				1.97						2341360
		4				1.92						1981599
		5				1.95						2184229
		6				1.92						2010127
		Mean				1.94						2119796

 

* = The loading level is considered to be not biologically significant, due to an inhibition ≤ 7%

Section-by-Section and Average Specific Growth Ratesof the Control Group (0 – 72 hours)

	Replicate No.	Specific Growth Rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.71	2.49	1.61	1.94	0.483	24.9	24.4
	2	1.60	2.45	1.76	1.94	0.451	23.3
	3	1.69	2.48	1.75	1.97	0.439	22.3
	4	1.52	2.55	1.68	1.92	0.555	28.9
	5	1.74	2.50	1.62	1.95	0.476	24.4
	6	1.75	2.42	1.60	1.92	0.436	22.7
				Mean	1.94
				SD ±	0.02
				CV [%]	1.04

 

SD = Standard deviation    CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, HOSTAGEL HT 300 was found to slightly inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the nominal loading of the test item): The EL50-values for both inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were > 100 mg/L. The NOEL-values for inhibition of growth rate and yield after 72 hours were 100 mg/L and < 100 mg/L, respectively. All effect values given are based on the nominal test item loading of HOSTAGEL HT 300.

Executive summary:

The toxicity of HOSTAGEL HT 300 (Batch no.:DEA14F316) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3 from 2016 -03 -14 to 2016 -03 -18 with the definitive exposure phase from 2016 -03 -15 to 2016 -03 -18,at the test facility. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6305 cells/mL. Based on a preliminary range finding test a water accommodated fraction with a nominal concentration of 100 mg/L was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

No analytical determination was carried out since no suitable method for the determination of the test item was available.

 

All effect values given are based on the nominal loading level of the test item HOSTAGEL HT 300.

 

NOEL, LOEL and EL-values of HOSTAGEL HT 300(0 - 72 hours)

based on nominal test item loading [mg/L]          

      

	Growth Rate Inhibition nominal test item loading [mg/L]
NOEL	100
LOEL	> 100
ErL10	> 100
ErL20	> 100
ErL50	> 100
	Yield Inhibition nominal test item loading [mg/L]
NOEL	< 100
LOEL	100
EyL10	< 100
EyL20	< 100
EyL50	> 100