reaction mass of diethyl (E)-2-methylbut-2-enedioate and diethyl (Z)-2-methylbut-2-enedioate

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:

2015-02-26 till 2015-04-10

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)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentration levels and the control were analytically verified after 0, 48 and 72 hours
Separate replicates for the test item analysis at the beginning of the exposure were prepared without algae. For the test item analysis after 48 hours separate samples were prepared with algae at the beginning of the exposure and incubated under test conditions. For the test item analysis after 72 hours separate replicates were prepared with and without algae at the beginning of the exposure and incubated under test conditions. Additionally the adsorption of the test item on the algae was analysed after 72 hours.
- Sample storage conditions before analysis: at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis (on an autosampler).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Each test concentration was prepared with dilution water (according to the guidline)

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÄK, SAG 61.81
- Source: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Method of cultivation: Nutrient medium Z according to Lüttge et al. (1994)

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE • m^-2 • s^-1 for 24 hours per day
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

none

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

22- 23°C, mean value: 22.5

pH:

8.04 - 9.29

Nominal and measured concentrations:

Nominal: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
time weight average: 0.704 - 2.28 - 7.45 - 24.8 - 82.5 mg/L
The measured concentrations of the test item in the fresh media (0 h) were in the range of 91 to 95 % of the nominal values at all tested concentration levels. The measured concentrations of the test item in the media after 48 hours were in the range of 70 to 79 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 42 to 78 % of the nominal values of all concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks
- Type: closed
- Material, size, headspace, fill volume: volume: 119 mL, with aluminium tops with PTFE seals
- Aeration: no
- Initial cells density: Nominal: approximately 5 x 103 - 104 cells/mL
Actual: 5526 cells/mL
- Control end cells density: 578042
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Culture medium: Nutrient medium Z accoridung to Lüttge et al. (1994)

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: According to OECD guideline with additional NaHCO3 (250mg/L) and MES monohydrate (2665.6 mg/L) to enable growth under conditions without headspace
- pH: 8.1 ± 0.2
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH and temperature were measured after 0 hours and 72 hours.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: 61 - 72.5 µE m-2 s-1, mean: 67.5 µE m-2 s-1
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: cell density was measured daily via Chlorophyll a- fluorescence, excitation at 436 nm, emission at 685 nm
- Chlorophyll measurement: Alga cells were evaluated microscopically at the start and end of the incubation period. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10
- Range finding study: yes
- Test concentrations: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

45.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

15.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.704 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13.9 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.51 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.704 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid: yes
- EC50(growth rate): 0.774, valid range 0.821 ± 0.388
- EC50(yield): 0.502, valid range 0.429 ± 0.261

Reported statistics and error estimates:

The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and Dunnett’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

Table 1: Measured Concentrations and Percentage of the Nominal Concentration of the test substance in Fresh Medium (0 hours) and Old Medium (72 hours)

   

Sampling date	2015-04-07 fresh medium without algae 0h		2015-04-09 old medium with algae 48h		2015-04-10 old medium with algae 72h
Nominal test item concentration [mg/L]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]	Time weighted mean [mg/L]
100	91.2	91	78.5	79	78.3	78	82.5
31.6	29.2	92	24.1	76	19.3	61	24.8
10.0	9.45	95	7.41	74	4.71	47	7.45
3.16	2.90	92	2.25	71	1.48	47	2.28
1.00	0.914	91	0.703	70	0.420	42	0.704
Control	< SysQL		< SysQL		< SysQL

Meas. conc.: Measured concentration of the test item (dilution factor taken into account)

%: Percent of the nominal concentration of the test item

SysQL: System Quantification Limit (0.1 mg/L test item)

Table 2: Microscopic evaluation of the cells at the start and end of exposure (Cell Densities)

Time weight average test item concentration [mg/L]	Replicate No.	Cell density [cells/mL]
		0h	24h	48h	72h
82.5	1	5526	7138	8944	13962
	2	5526	6881	7389	13334
	3	5526	7737	9603	13090
	Mean	5526	7252	8645	13462
24.8	1	5526	11058	25480	232905
	2	5526	10490	36591	203646
	3	5526	10772	19145	230992
	Mean	5526	10773	27072	222514
7.45	1	5526	17731	79429	383683
	2	5526	18813	97514	361261
	3	5526	17565	83703	407047
	Mean	5526	18036	86882	383997
2.28	1	5526	23508	128234	467227
	2	5526	20789	123659	493694
	3	5526	24423	131860	489147
	Mean	5526	22907	127918	483356
0.704	1	5526	26487	186370	569680
	2	5526	26528	180373	526466
	3	5526	26227	185413	574133
	Mean	5526	26414	184052	556760
Control	1	5526	30742	185363	613270
	2	5526	20105	187326	506427
	3	5526	25653	203153	532894
	4	5526	19653	203254	648675
	5	5526	28296	209206	586865
	6	5526	30256	210492	580123
	Mean	5526	25784	199799	578042

Table 3: Evaluation after 72 hours. Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Time weight average test item concentration [mg/L]	Replicate No.	Growth rate [d-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
82.5	1	0.390	80	8436	99
	2	0.294	81	7808	99
	3	0.287	81	7564	99
	Mean	(+) 0.297	81	(+) 7936	99
24.8	1	1.25	19	227379	60
	2	1.20	22	198120	65
	3	1.24	20	225466	61
	Mean	(+)1.23	21	(+) 216988	62
7.45	1	1.41	9	378157	34
	2	1.39	10	355735	38
	3	1.43	7	401521	30
	Mean	(+)1.41	9	(+) 378471	34
2.28	1	1.48	5	461701	19
	2	1.50	3	488168	15
	3	1.49	4	483621	16
	Mean	(+)1.49	4	(+) 477830	17
0.704	1	1.55	0	564154	1
	2	1.52	2	520940	9
	3	1.55	0	568607	1
	Mean	(-) 1.54	1	(-) 551234	4
Control	1	1.57		607744
	2	1.51		500901
	3	1.52		527368
	4	1.59		643149
	5	1.56		581339
	6	1.55		574597
	Mean	1.55		572516

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 (2011) and Council Regulation (EC) No 761/2009 C.3 at the test facility. After 72 hours the NOEC was determined to be 0.704 mg/L, the ErC10 was determined to be 15.3 mg/L and the ErC50 was determined to be 45.1 mg/L.

Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201.

The study was conducted under static conditions with an initial cell density of 5526 cells/mL. With regard to the volatility of the test item, glass flasks without headspace were used to reduce losses of the test item. Five concentrations were tested in a geometrical series with a dilution factor of V(10) (nominal): 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L, corresponding to the time weight average test item concentrations: 0.704 - 2.28 - 7.45 - 24.8 - 82.5 mg/L.

The concentrations of the test item were analytically verified by HPLC-DAD.

The measured concentrations of the test item in the fresh media (0 h) were in the range of 91 to 95 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 42 to 78 % of the nominal values of all concentrations.

After 72 hours the ErC10 was determined to be 15.3 mg/L (TWA) and the ErC50 was determined to be 45.1 mg/L (TWA).

Description of key information

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 (2011) and Council Regulation (EC) No 761/2009 C.3 at the test facility. After 72 hours the ErC10 was determined to be 15.3 mg/L and the ErC50 was determined to be 45.1 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

45.1 mg/L

EC10 or NOEC for freshwater algae:

15.3 mg/L

Additional information

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201.

The study was conducted under static conditions with an initial cell density of 5526 cells/mL. With regard to the volatility of the test item, glass flasks without headspace were used to reduce losses of the test item. Five concentrations were tested in a geometrical series with a dilution factor of V(10) (nominal): 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L, corresponding to the time weight average test item concentrations: 0.704 - 2.28 - 7.45 - 24.8 - 82.5 mg/L.

The concentrations of the test item were analytically verified by HPLC-DAD.

The measured concentrations of the test item in the fresh media (0 h) were in the range of 91 to 95 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 42 to 78 % of the nominal values of all concentrations.

After 72 hours the ErC10 was determined to be 15.3 mg/L (TWA) and the ErC50 was determined to be 45.1 mg/L (TWA).