Ethoxycarbonylmethyl ethyl phthalate

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:

July 2019 - August 2019

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentration of the test item was measured at each test concentration respectively the control at the beginning and end of the test in triple determination. Additionally two test solutions without algae were prepared for the analytical determination.
The concentration of the test item was measured in each tested concentration at the beginning and the end of the test in double determination. Measurements of the control at the beginning and the end of the test were done.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of the test item of 111 mg/l was prepared in growth medium. The sample was weighed using a weighing dish. The solution was stirred overnight at room temperature afterwards. The test solutions were prepared by adding growth medium and inoculum culture to the stock solution of the test item.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: SAG 86.81
- Method of cultivation: A preparatory culture in the growth medium was prepared 4 days before start of the test. The preparatory culture was incubated under the same conditions as the test cultures. The algae showed a normal and healthy appearance under the microscope after the incubation. The preparatory culture was used as inoculum culture after adjusting the cell concentration with growth medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.3 to 23.0 °C

pH:

7.6 - 8.6

Nominal and measured concentrations:

Nominal test concentrations were 6.25, 12.5, 25.0, 50.0 and 100 mg/L. The factor between concentrations was 2.

Details on test conditions:

TEST SYSTEM
- Test vessel: cuvettes (PS) with lids
- Fill volume: 3 mL
- Initial cells density: 3.9 x 10exp3 cell/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: OECD TG 201 growth medium

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: pH-values of the solutions were measured at the beginning and end of the test for each test concentration.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 7490 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: algal biomass in each test vessel was measured as chlorophyll fluorescence daily

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 100 mg/L, 10 mg/L, 1.0 mg/L, 0.1 mg/L and 0.01 mg/L
- Results used to determine the conditions for the definitive study: The expected ErC50-value will be between 10 and 100 mg/l.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

25.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 23.5-28.3 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 27.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

10.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI: 7.9-13.7 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 27.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Results with reference substance (positive control):

The determined ErC50 of 0.78 mg/l lies within the range from the inter laboratory test to ISO 8692 (0.84 ± 0.24 mg/l).

Reported statistics and error estimates:

From the determined effects the EC50-, EC20- and EC10-values of the test item were calculated by non-linear regression. The calculation was carried out with the software SigmaPlot 13.

Results of analysis

The recovery of the nominal concentration in the individual concentrations at the beginning was between 25 and 50 %. The low recovery may be due to the use of a weighing dish in the preparation of the stock solution. It is also possible that other ad- or absorption effects or the lifetime of the sample until processing may have played a role.

The recovery of the initial concentration at the end of the test was between 6 and 51 %. Thus, the substance was not stable over the test period or further ad- or absorption effects occurred. This cannot be conclusively clarified.

The test organism had an influence on the recovery, since the solutions without algae showed a higher recovery between start and end value than the solutions with algae.

Therefore, a mean test concentration was calculated over the test period to determine the dose-response ratio. The average of the initial and final concentrations for each test solution was used. The concentration at the end of the test was calculated from the mean recovery in the solutions without algae (59.3 %) multiplied by the determined initial values.

 

Nominal and determined concentrations of the test item in the control and each test concentration.

Nominal concentration ofEpeg	Determined concentration of Epeg		Mean concentration of Epeg determined over the test period
	at 0 hours	at 72 hours
[mg/l]	[mg/l]	[mg/l]	[mg/l]
0 (control)	1.2	0.0	0.9
6.25	3.1	0.2	2.5
12.5	3.1	0.5	2.5
25	8.7	1.2	6.9
50	15.7	4.6	12.5
100	34.2	17.4	27.2
0 (blank)^1)	0.3	0.2	0.2
12.5^1)	3.7	2.2	3.0
100^1)	34.0	20.7	27.1

¹⁾test solutions without algae

 

Results of the test:

The growth curve of the control group shows that the controls have largely grown exponentially over the course of the test. This can be seen from the almost straight course of the curve with logarithmic application of the biomass (here as fluorescence). The growth of the controls in the last 24 hours of the test duration was not substantially reduced.

The control cultures achieved over the whole test duration an average growth rate of 1.6 d^-1. This corresponds to an increasing biomass by a factor of 119.

The mean coefficient of variation of the daily growth rates was 6.5 %, the coefficient of variation over the whole test period was 0.9 %.

 

Percent inhibition of the growth rate for the treatment replicates after 72 hours:

Epeg	Parallel	Inhibition of the growth rate		Inhibition of the yield
Conc.^1)		[%]		[%]
[mg/l]		Single value	Mean	Single value	Mean
	a	-0.6		-3.1
2.5	b	-0.5	-0.4	-2.3	-1.9
(6.25)	c	-0.1		-0.4
	a	-1.5		-7.3
2.5	b	-1.1	-1.3	-5.2	-6.2
(12.5)	c	-1.2		-6.0
	a	0.1		0.5
6.9	b	-0.7	-0.1	-3.5	-0.4
(25)	c	0.4		2.0
	a	3.2		14.3
12.5	b	4.0	4.2	17.6	18.2
(50)	c	5.3		22.6
	a	9.7		37.6
27.2	b	11.2	10.9	42.0	40.9
(100)	c	11.6		43.0

¹⁾determined (nominal) concentration

 

Calculated effect concentrations after 72 hours:

	Average specific growth rate			Yield
	Concentration	95% Confidence Limits		Concentration	95% Confidence Limits
	[mg/l]	lower	upper	[mg/l]	lower	upper
EC10	25.6	23.5	28.3	10.6	7.9	13.7
EC20	>27.2	-	-	15.9	13.0	18.6
EC50	>27.2	-	-	>27.2	27.8	-

 

Validity criteria fulfilled:

yes

Conclusions:

The test item caused an inhibition of the average specific growth rate of 10.9 % in the highest test concentration of 27.2 mg/l after 72 hours. The ErC50-value was therefore > 27.2 mg/l. The ErC10-value was 25.6 mg/l.

Executive summary:

The toxicity of Ethylphthalyl ethyl glycolate to the Freshwater Alga Desmodesmus subspicatus was tested according to the OECD Guideline 201.

The test item caused an inhibition of the average specific growth rate of 10.9 % in the highest test concentration of 27.2 mg/l after 72 hours. The ErC50-value was therefore > 27.2 mg/l. The ErC10-value was 25.6 mg/l.

Description of key information

The toxicity of Ethylphthalyl ethyl glycolate to the Freshwater Alga Desmodesmus subspicatus was tested according to the OECD Guideline 201.

The test item caused an inhibition of the average specific growth rate of 10.9 % in the highest test concentration of 27.2 mg/l after 72 hours. The ErC50-value was therefore > 27.2 mg/l. The ErC10-value was 25.6 mg/l.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

25.6 mg/L