Ethanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanamine

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:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanamine

EC Number:

948-066-8

Molecular formula:

C16H36NO6P C24H52NO8P C24H53NO11P2 C32H68NO10P C8H17N H3O4P

IUPAC Name:

Ethanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanamine

Test material form:

liquid: viscous

Specific details on test material used for the study:

Batch/Lot number: 0002293298
Appearance: High-viscous, colourless to yellowish liquid
Purity: Considered as 100%
Expiry date: 22 November 2019
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials will be applied to assure personnel health and safety.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentration of the test item was measured at the test concentration at the start and at the end of the experiment. Sample from the control was taken for analysis at the beginning and at the end of the experiment.
All samples will be analysed directly after sampling.

Test solutions

Vehicle:

no

Details on test solutions:

A stock solution with a nominal concentration of 20 mg/L was prepared with direct addition of the test item, mixed into the test medium (OECD medium) using ultrasonic bath (approximately 3 minutes). The test solutions were prepared by appropriate diluting of this stock solution just before treatment (see Table 2 below).

Test organisms

Test organisms (species):

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

Details on test organisms:

Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)

Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD No. 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd.

Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.

Breeding conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines. The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture will be prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they will be discarded.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation

Test conditions

Test temperature:

22.0°C - 22.3°C

pH:

7.36 – 8.95

Details on test conditions:

The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 7502 lux (equivalent to ~101 E/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed  15 % and therefore provided equal conditions for each test vessel.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 14.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 2.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Yield

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 7.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Details on results:

AVERAGE SPECIFIC GROWTH RATES


The results of the statistical evaluation (based on Bonferroni t-Test; =0.05) show that the 0-72 hours average specific growth rate was statistically significantly different from the untreated control value in the measured concentration range of 5 - 20 mg/L (nominal), accordingly the No Observed Effect Concentration (NOEC) was determined as 2.5 mg/L (nominal).
The 72 h ErC50 value was determined [by Probit analysis (TOXSTAT software)] as 14.9 mg/L (95 % confidence limits: 12.9 – 17.1 mg/L).

AREAS UNDER THE GROWTH CURVES


The results of the statistical evaluation (based on Bonferroni t-Test; =0.05) show that the 0-72 h areas were statistically significantly different from the untreated control value at the measured concentration range of 5 – 20 mg/L (nominal), accordingly the No Observed Effect Concentration (NOEC) was determined as 2.5 mg/L (nominal).
The 72 h EbC50 value was determined [by Probit analysis (TOXSTAT software)] as 7.3 mg/L (95 % confidence limits: 6.6 – 8.1 mg/L).

YIELD


The results of the statistical evaluation (based on Bonferroni t-Test; =0.05) show that the 0-72 h yield was statistically significantly different from the untreated control value at the tested concentration range of 5 - 20 mg/L (nominal), accordingly the No Observed Effect Concentration (NOEC) was determined as 2.5 mg/L (nominal).

The 72 h EyC50 value was determined [by Probit analysis (TOXSTAT software)] as 7.0 mg/L (95 % confidence limits: 6.4 – 7.7 mg/L).

Results with reference substance (positive control):

The 72h ErC 50: 0.87 mg/L, (95 % confidence limits: 0.80 – 0.95 mg/L)
The 72h EbC 50: 0.62 mg/L, (95 % confidence limits: 0.57 – 0.68 mg/L)
The 72h EyC 50: 0.52 mg/L, (95 % confidence limits: 0.48 – 0.57 mg/L)

These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Any other information on results incl. tables

Table 5: Growth Rates (m) and Percentage Inhibition ofmduring the Test Period

Nominal concentration	Growth rate (m) and % inhibition ofm
	0–24 h		0–48 h		0–72 h
[mg/L]	m	%	m	%	m	%
Control	0.0553^+	0.0	0.0618	0.0	0.0595	0.0
1.25	0.0569	-2.8	0.0620	-0.3	0.0597	-0.4
2.5	0.0578	-4.4	0.0586	5.2	0.0585	1.6
5	0.0569	-2.8	0.0527*	14.7	0.0556*	6.5
10	0.0458	17.2	0.0415*^+	32.9	0.0411*^+	30.9
20	0.0289*	47.8	0.0249*	59.7	0.0211*^+	64.5

*: statistically significantly different compared to the control values (Bonferroni t-Test ;a= 0.05)

+: at these values the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

Table 6: Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Nominal concentration	Area under the Growth Curves (A) and Percentage Inhibition of A
	0–24 h		0–48 h		0–72 h
[mg/L]	A	%	A	%	A	%
Control	34.0	0.0	290.0	0.0	1368.0	0.0
1.25	36.0	-5.9	296.0	-2.1	1392.0	-1.8
2.5	36.0	-5.9	260.0	10.3	1248.0	8.8
5	36.0	-5.9	212.0*	26.9	996.0*	27.2
10	24.0	29.4	124.0*	57.2	420.0*	69.3
20	12.0*	64.7	52.0*	82.1	124.0*	90.9

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Table 7: Yield (Y) and Percentage Inhibition of Y during the Test Period

Nominal concentration		Yield and Inhibition
		0–72 h
[mg/L]		Y		%
Control		71.3		0.0
1.25		72.7		-1.9
2.5		66.7		6.5
5		53.7*		24.8
10		18.3*		74.3
20		3.7*		94.9

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of Ethanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanamine test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours.

The biological results are summarised in the following table:
Table 8: Influence of Ethanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanamine on the Growth of Pseudokirchneriella subcapitata
Parameter
(0-72 h) Growth rate (r)
[mg/L] Yield (y)
[mg/L] Biomass (b)
[mg/L]
Calculation based on measured geometric mean concentrations
EC50 14.9 7.0 7.3
95 % conf. limits 12.9 – 17.1 6.4 – 7.7 6.6 – 8.1
NOEC 2.5 2.5 2.5
LOEC 5.0 5.0 5.0

Executive summary:

The effect of test item was assessed on algal growth using the unicellular green algaPseudokirchneriella subcapitata(Selenastrum capricornutum), over an exposure period of 72 hours.

As significant toxic responsewas observed at the examined concentration levels during the preliminary range-finding test, fivetest concentrations in a geometric series (factor 2.0) and one controlwere tested in the main experiment.

The concentrations oftest item used in the main experimentwere:1.25, 2.5, 5, 10 and20 mg/L.

Measured values of test item could only be quantified at concentrations of10 and 20 mg/L. The concentration of 5 mg/L was below theLimit of Quantificationat the start of the experiment, but at the end of the experiment it could be measured (5.5 mg/L). The two lowestconcentrations were below quantification limit, thus the geometric mean could not be correctly calculated. All the results/conclusions in this report are expressed as the nominal concentrations, which is considered to be the validapproach for a UVCB substances (Unknown or Variable Composition, Complex Reaction Products and Biological Materials) based on OECD No. 23 guidance.

The test design included three replicates at each test concentration and six replicates for the untreated controls.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (a= 0.05) by TOXSTAT software.

TheE_rC₅₀, E_bC₅₀and E_yC₅₀values of the test item and their confidence limits were calculated using Probit analysisby TOXSTAT software.

 

The biological results are summarised in the following table:

 

Table 1:  Influence ofEthanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanamineon the Growth ofPseudokirchneriella subcapitata

Parameter (0-72 h)	Growth rate (r) [mg/L]		Yield (y) [mg/L]	Biomass (b) [mg/L]
		Calculation based on measured geometric mean concentrations
EC50	14.9		7.0	7.3
95 % conf. limits	12.9 – 17.1		6.4 – 7.7	6.6 – 8.1
NOEC	2.5		2.5	2.5
LOEC	5.0		5.0	5.0