1,1-diethoxyethane

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 2018-06-22 to 2018-09-21

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

Remarks:

GC-MS

Vehicle:

no

Details on test solutions:

A stock solution with a nominal test item concentration of 100 mg/L was freshly prepared with dilution water prior to the start of exposure. The stock solution was shaken manually.
GC-MS/MS conditions:
Injector: Split 20
Injector temperature: 200 °C
Agitator: 55 °C
Incubation time: 3 min
Extraction time: 6 min
Desorption time: 60 s
Carrier gas: Ramped Pressure equalling a constant flow of approximately 1 mL/min
Retention time: Approx. 3.1 min (test item), 4 min (ISTD)
GC oven programme:
50 °C - hold 1.5 min - 75 °C - heating rate 10 °C/min - hold 0 min - 200 °C - 40 °C/min - hold 2 min
Conditions of detection:
Source Temperature: 250 °C
Ionisation mode: Electron impact ionization (El), positive
Scan mode: Centroid
Scan method: MS/MS

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts to 2567 - 5130 lux for 24 hours per day.
- Culture medium: Nutrient medium Z according to Lüttge et al. (1994).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.5 - 23.5 °C

pH:

start: 8.08 - 8.24
end: 9.35 - 9.43

Nominal and measured concentrations:

Nominal concentrations: 0.0977 - 0.391 - 1.56 - 6.25 - 25.0 - 100 mg/L
Measured concentrations: 0.163 - 0.445 - 1.67 - 6.09 - 22.8 - 94.4 mg/L (initial)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks
- Type: closed
- Material, size, headspace, fill volume: Sterile headspace flasks, volume: 59 mL, with aluminium tops with PTFE seals, test volume: 59 mL
- Aeration: none
- Initial cells density: 6442 cells/mL (approximately 5 x 103 -104 cells/mL)
- Control end cells density: 80-fold the initial cell density
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD 201
- Culture medium different from test medium: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: none
- Photoperiod: 24 hours/day light
- Light intensity and quality: 5224 to 6664 lux (mean 6069 lux), corresponding to 60 to 120 µE/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Microscopic evaluation of the cells was carried out at the start and end of the incubation. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 4
- Range finding study: yes
- Test concentrations: 1.00 - 10.0 - 100 mg/L
- Results used to determine the conditions for the definitive study: 9 % inhibition was observed at conc. level 1 and 100 mg/L. 10 % inhibition was observed at conc. level of 10 mg/L. The yield inhibition varied between 34 and 37 %.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 94.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 94.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 94.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 94.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 94.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.163 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.163 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Results with reference substance (positive control):

In the last reference test with the reference item was determined that the ErC50 with headspace is 1.09 mg/L (95 % CI: 0.842 - 1.31 mg/L) and without headspace is 0.2444 mg/L (95 % CI: 0.175 - 0.350 mg/L).

Table 1. Evaluation of growth rates and yield after 72 hours

Initial measured test item concentration (mg/L)	Replicate No.	Growth rate (1/d)	Growth rate inhibition (%)	Yield (1/d)	Yield inhibition (%)
94.4	1 2 3	1.39 1.38 1.42	5 5 3	407051 402582 448037	20 21 12
	Mean	(+)*        1.40	4	(+) 419223	18
22.8	1 2 3	1.28 1.30 1.38	13 11 6	290944 309985 394886	43 39 22
	Mean	(+)*        1.32	10	(+) 331938	35
6.09	1 2 3	1.42 1.25 1.34	3 14 9	453076 268726 347073	11 47 32
	Mean	(+)*        1.34	8	(+) 356292	30
1.67	1 2 3	1.34 1.41 1.42	8 4 3	348042 433142 443866	32 15 13
	Mean	(+)*        1.39	5	(+) 408350	20
0.445	1 2 3	1.39 1.33 1.40	5 9 4	403972 338484 422765	21 33 17
	Mean	(+)*        1.37	6	(+) 388407	24
0.163	1 2 3	1.42 1.39 1.37	2 5 6	455608 404047 390716	10 21 23
	Mean	(+)*        1.39	4	(+) 416790	18
Control	1 2 3 4 5 6	1.45 1.49 1.47 1.45 1.43 1.47		490934 549074 527700 497165 467102 518291
	Mean	1.46		508378

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

* = Since inhibition values were <=10 % and showed no concentration dependent correlation, growth rate inhibition was considered to be not significant by expert judgement.

Table 2. Measured concentrations and percentage of the nominal conc. of the test item

Sampling date	Fresh media, 0 hours		Old media, 72 hours
Nominal test item conc. (mg/L)	Measured conc. (mg/L)	%	Measured conc. (mg/L)	%
100	94.4	94	95.6	96
25.0	22.8	91	23.7	96
6.25	6.09	97	6.03	96
1.56	1.67	107	1.73	111
0.391	0.445	114	0.439	112
0.0977	0.163	167	0.144	147
Control	<LOQ		<LOQ

LOQ = 0.1 µg/L

Validity criteria fulfilled:

yes

Conclusions:

The 72-h ErC50 and ErC10 based on the growth rate for the test item were determined to be >94.4 mg/L (initial measured conc.).

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to OECD 201 and Council Regulation (EC) No. 266/2016, C.3. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. A stock solution with a nominal test item concentration of 100 mg/L was freshly prepared with dilution water prior to the start of exposure. The stock solution was shaken manually. The study was conducted under static conditions with an initial cell density of 6442 cells/mL. With regard to the volatility of the test item, glass flasks without headspace were used to reduce losses of the test item. Six concentrations were tested in a geometrical series with a dilution factor of 4 (nominal): 0.0977 - 0.391 - 1.56 - 6.25 - 25.0 - 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The test media were clear and the test item was dissolved throughout the test period. The concentrations of the test item in the solutions and the control were analytically verified via GC-MS at the start of exposure (0 h) and at the end of exposure (72 hours) period. The measured concentrations of the test item in fresh media were in the range of 91 – 167 % of the nominal values at the start of the exposure (0 hours) and 95 – 147 % after 72 hours at the end of exposure. The ErC50 and ErC10 based on the growth rate were determined to be >94.4 mg/L (initial measured conc.). The EyC50 based on the yield was determined to be >94.4 mg/L (initial measured conc.). An EyC10 was not determinable. Respectively, the NOEC based on growth rate was >=94.4 mg/L and based on yield was <0.163 mg/L.

Description of key information

The ErC50 and ErC10 based on the growth rate were determined to be >94.4 mg/L (initial measured conc.) after 72-h exposure to the test item.

Additional information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to OECD 201 and Council Regulation (EC) No. 266/2016, C.3 (reference 6.1.5-1). The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. A stock solution with a nominal test item concentration of 100 mg/L was freshly prepared with dilution water prior to the start of exposure. The stock solution was shaken manually. The study was conducted under static conditions with an initial cell density of 6442 cells/mL. With regard to the volatility of the test item, glass flasks without headspace were used to reduce losses of the test item. Six concentrations were tested in a geometrical series with a dilution factor of 4 (nominal): 0.0977 - 0.391 - 1.56 - 6.25 - 25.0 - 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The test media were clear and the test item was dissolved throughout the test period. The concentrations of the test item in the solutions and the control were analytically verified via GC-MS at the start of exposure (0 h) and at the end of exposure (72 hours) period. The measured concentrations of the test item in fresh media were in the range of 91 – 167 % of the nominal values at the start of the exposure (0 hours) and 95 – 147 % after 72 hours at the end of exposure. The ErC50 and ErC10 based on the growth rate were determined to be >94.4 mg/L (initial measured conc.). The EyC50 based on the yield was determined to be >94.4 mg/L (initial measured conc.). An EyC10 was not determinable. Respectively, the NOEC based on growth rate was >= 94.4 mg/L and based on yield was <0.163 mg/L.