2,2'-[oxybis(2,1-ethanediyloxy)]bisacetic acid

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:

from 2018-02-12 to 2018-05-31

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and Assessment No. 23, Paris September 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Lot/batch No(s): 03/2016_R_JM

Analytical monitoring:

yes

Details on sampling:

Three parallel samples were measured from each test concentration and four/six replicates from the control.

Vehicle:

no

Details on test solutions:

A stock solution of 100 mg/L (nominal concentration) was prepared by dissolving 0.09 g of the test item in 900 mL OECD Medium. The pH value (4.42) of this stock solution was adjusted to the pH value of the OECD medium (8.12) (according to OECD 23). The adjusted pH level of the stock solution was 8.40. Further test solutions were prepared by appropriate dilution of this stock solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Source: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): 4 days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.7 – 22.8 °C

pH:

8.08 - 8.89

Nominal and measured concentrations:

nominal concentrations: 100, 50, 25, 12.5, 6.25 mg/L
measured concentrations: 101.2, 51.2, 25.6, 12.7, 6.31 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: glass, ~250 mL volume, 100 mL test medium
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 126.50 x 10^4 cells/mL
- No. of organisms per vessel: 0.1 mL algal biomass (10^7 algal cells per 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: OECD Medium prepared according to OECD 201

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: continuously illuminated
- Light intensity and quality: 8053 lux

TEST CONCENTRATIONS
- Range finding study; yes, 2 tests, with and without pH adjustment
- Test concentrations: 0.1, 1, 10, 100 mg/L

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: microscope with counting chambe at 24, 48 and 72 hours

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CI: 1.016 – 19.675 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Unusual cell shape: No morphological abnormalities were observed in the control and in the concentrations of 6.25, 12.5, 25.0 mg/L during the experiment. At the concentrations of 50 and 100 mg/L some swollen cells were observed together with normal cells at 24, 48 and 72 hours.

Results with reference substance (positive control):

The date of the last study with the reference item Potassium dichromate was: 26 February – 01 March 2018. The following results were obtained:
The 72h ErC50: 1.08 mg/L, (95 % confidence limits: 0.79 – 1.51 mg/L)
The 72h EyC50: 0.61 mg/L, (95 % confidence limits: 0.49 – 0.76 mg/L)

Pre-experiments

In the preliminary tests, the test item showed 100 % growth inhibition at 100 mg/L tets concentration when the pH of the test item solution was not adjusted. After adjustment of the pH, the inhibition at 100 mg/L concentration level was only 3.80 %. In the second preliminary test, performed with adjusted pH, the inhibition of the algae was at a maximum of 3.29 % at treatment level of 100 mg/L.

Validity criteria

- Cell density in the control cultures increased by a factor of more than 16 (by a factor of 79.67) within 72 hours.

- The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %.

- CV for section-by-section growth rate day 0-1: 21.73 %

- CV for section-by-section growth rate day 1-2: 12.59 %

- CV for section-by-section growth rate day 2-3: 3.86 %

The mean coefficient of variation for section-by-section specific growth rates: 12.73 %.

- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % in test.

- CV for average specific growth rate day 0-3: 2.91 %

All validity criteria were met, therefore, the study is considered as valid.

Analytical results

The measured concentration of the test item in the test solutions wasin the range of 97 – 98 % at the start and 101 – 102 % at the end of the test. Test item was not detected in the control group. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the biological results was based on the nominal concentration values.

Biological results

The test item had no statistically significant inhibitory effect on the growth of Raphidocelis subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield at the concentrations of 6.25, 12.5, 25.0 mg/L compared to the control.

The ErCx and EyC50 values of the test item were determined and given directly from the raw data.

The EyC10 and EyC20 values of the test item were calculated using Probit.

Accordingly, the 72-hour NOEC based on growth rate and yield was determined to be 25.0 mg/L and the LOEC as 50.0 mg/L.

In this 72-h algal growth inhibition test, the obtained results showed that the test item has statistically significant toxic effects on the growth of Raphidocelis subcapitata at the concentrations of 50.0 and 100.0 mg/L.

Table 1: Summary of biological endpoints

Endpoints (0 - 72 h)	Growth rate (µ) [mg/L]	Yield (y) [mg/L]
EC10	> 100	9.91
95 % conf. limits		(1.016 – 19.675)
EC20	> 100	43.381
95 % conf. limits		(22.556 – 135.092)
EC50	> 100	> 100
NOEC	25.0	25.0
LOEC	50.0	50.0

 

Table 2. Growth Rates (µ) and percentage inhibition of µ during the test period

Nominalconcentration (mg/L)	Growth Rates (µ) and % Inhibition of µ
	0–24 h		0–48 h		0–72 h
	µ	%	µ	%	µ	%
Untreated control	0.0401	0.0	0.0508	0.0	0.0607	0.0
6.25	0.0345	14.0	0.0486	4.3	0.0590	2.8
12.5	0.0345	14.0	0.0517	-1.7	0.0596	1.9
25.0	0.0345	14.0	0.0512	-0.7	0.0598	1.5
50.0	0.0193	52.0	0.0465	8.5	0.0568	6.5
100.0	0.0000	100.0	0.0457	10.1	0.0560	7.8

Table 3. Yield (y) and percentage inhibition of y during the test period

Nominalconcentration (mg/L)	Yield (y) and % Inhibition of y
	0–72 h
	y	%
Untreated control	78.67	0.0
6.25	69.00	12.3
12.5	72.33	8.1
25.0	73.00	7.2
50.0	58.67	25.4
100.0	55.33	29.7

Table 4. The section-by-section specific growth rates in the control cultures

Section-by-Section Growth Rate
Expressed in hours	0–24 h	24-48 h	48-72 h
	0.0401	0.0615	0.0805
Expressed in days	0-1 day	1-2 day	2-3 day
	0.9635	1.4766	1.9313

Validity criteria fulfilled:

yes

Conclusions:

In this 72-h algal growth inhibition test, the obtained results showed that the test item has statistically significant toxic effects on the growth of Raphidocelis subcapitata at the concentrations of 50.0 and 100.0 mg/L. The EC10 and EC50 based on growth rate were determined to be > 100 mg/L, respectively. The NOEC was determined to be 25 mg/L.

Executive summary:

The effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata was determined according to OECD 201, Regulation (EC) No. 226/2016 Method C.3 and EPA OPPTS 850.5400 under GLP. The study was conducted under static conditions with an initial cell density of 1x10^4 cells/mL for a period of 72 hours. Based on a series of preliminary tests it was concluded that the pH of the test item stock solution has to be adjusted to the pH of the dilution water (OECD Medium). In these preliminary tests the test item showed inhibitory effects on the tested algae, therefore, a concentration range of6.25, 12.5, 25.0, 50.0 and 100 mg/L as nominal concentrations was tested in the main test.The measured concentrations of the test item at the start and at the end of the test were in the range of 100 - 102 % of the nominal concentration. As th measured concentrations did not deviate more than 20 % from the nominal concentrations, the biological results were based on the nominal concentrations. The test item showed statistically significant toxic effects on the growth of Raphidocelis subcapitata at the concentrations levels of 50 and 100 mg/L. The EC50-values for inhibition of growth rate (ErC50) and for inhibition of yield (EyC50) were >100 mg/L. The ErC10 and EyC10 values were determined to be >100 mg/L and 9.91 mg/L, respectively. The NOEC was determined to be 25 mg/L.

Description of key information

In a 72-h algal growth inhibition test, the obtained results showed that the test item has statistically significant toxic effects on the growth of Raphidocelis subcapitata at the concentrations of 50.0 and 100.0 mg/L. The EC10 and EC50 based on growth rate were determined to be > 100 mg/L, respectively. The NOEC was determined to be 25 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

25 mg/L

Additional information

The effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata was determined according to OECD 201, Regulation (EC) No. 226/2016 Method C.3 and EPA OPPTS 850.5400 under GLP. The study was conducted under static conditions with an initial cell density of 1x10^4 cells/mL for a period of 72 hours. Based on a series of preliminary tests it was concluded that the pH of the test item stock solution has to be adjusted to the pH of the dilution water (OECD Medium). In these preliminary tests the test item showed inhibitory effects on the tested algae, therefore, a concentration range of6.25, 12.5, 25.0, 50.0 and 100 mg/L as nominal concentrations was tested in the main test.The measured concentrations of the test item at the start and at the end of the test were in the range of 100 - 102 % of the nominal concentration. As th measured concentrations did not deviate more than 20 % from the nominal concentrations, the biological results were based on the nominal concentrations. The test item showed statistically significant toxic effects on the growth of Raphidocelis subcapitata at the concentrations levels of 50 and 100 mg/L. The EC50-values for inhibition of growth rate (ErC50) and for inhibition of yield (EyC50) were >100 mg/L. The ErC10 and EyC10 values were determined to be >100 mg/L and 9.91 mg/L, respectively. The NOEC was determined to be 25 mg/L.