Dimethyl (p-methoxybenzylidene)malonate

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, 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:

Determination of the test item
Analytical evaluation of the concentrations of the test item was carried out via HPLC-DAD from freshly prepared media after 0 hours and old test media after 72 hours of exposure. Samples for the test item analysis at the beginning of the exposure were taken from additionally prepared replicates with algae. For the test item analysis after 72 hours, samples were taken from the test replicates.

Vehicle:

no

Details on test solutions:

Stock solution, preparation of the test media
A stock solution of 50 mg/L was prepared with dilution water and stirred for 24 ± 1 hours (room temperature, 1100 rpm).

Test concentrations
6 concentrations were tested in a geometric series with a dilution factor of √10: 0.0632 - 0.200 - 0.632 - 2.00 - 6.32 - 20.0 mg test item/L.
The nominal concentrations were based on the results of a preliminary range finding test (non-GLP).

Control
Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

not specified

Test temperature:

min.: 21.0 max.: 23.0, mean value: 22.0 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
20.0 7.53 7.90
6.32 7.50 8.00
2.00 7.48 8.04
0.632 7.48 8.18
0.200 7.57 8.33
0.0632 7.75 8.40
Control 8.05 8.60

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

See window "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 4847 cells/mL
- Control end cells density: Mean 1251332 cells/mL
- 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:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m^-2 x s^-1, mean value: 68.2 µE x m^-2 x s^-1,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.632 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.963 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.506 - 1.67

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.72 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 1.93 - 3.49

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 7.77 - 11.3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.632 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 0.063 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.298 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: < 0.0632 - 0.541

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 0.816 - 1.47

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) 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):

The toxicity of potassium dichromate (SIGMA, batch number MKBQ9179V, purity 100.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of 72 hours from 2015-10-19 to 2015-10-22. The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.685 0.671 ± 0.401
95 % confidence interval 0.603 - 0.794
Yield Inhibition
EyC50 0.362 0.320 ± 0.139
95 % confidence interval 0.328 - 0.398
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals of the EC10-, EC20- and EC50-values was carried out using standard procedures.

NOEC and LOEC values
The NOEC / LOEC was determined by calculation of statistical significance of growth rate 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. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05. The normality test failed for yield. The highest concentration was eliminated to pass the normality test for yield.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Cell Densities

Nominal test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
20.0	1	4847	4866	6930	9523
	2	4847	5001	6627	5621
	3	4847	3452	8714	9215
	Mean	4847	4440	7424	8120
6.32	1	4847	18470	60802	186318
	2	4847	20293	75956	206464
	3	4847	17696	73579	184413
	Mean	4847	18820	70112	192398
2.00	1	4847	26085	98661	331095
	2	4847	23598	119933	364480
	3	4847	29948	139522	420455
	Mean	4847	26544	119372	372010
0.632	1	4847	31107	162881	779174
	2	4847	29645	166047	814435
	3	4847	32892	180844	839161
	Mean	4847	31215	169924	810923
0.200	1	4847	31280	164719	997859
	2	4847	26691	214950	1222750
	3	4847	30448	230411	1267055
	Mean	4847	29473	203360	1162555
0.0632	1	4847	29351	167240	957884
	2	4847	26821	179073	929839
	3	4847	29207	206513	1079686
	Mean	4847	28460	184275	989136
Control	1	4847	33190	235549	1581565
	2	4847	30342	219251	1123750
	3	4847	24656	238050	1378273
	4	4847	23391	205435	994636
	5	4847	31646	241937	1226887
	6	4847	31175	207427	1202883
	Mean	4847	29067	224608	1251332

Evaluation after 72 hours

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

 

Nominal test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
20.0	1		0.23	88		4676	100
	2		0.05	97		774	100
	3		0.21	88		4368	100
	Mean	(+)	0.16	91	(+)	3273	100
6.32	1		1.22	34		181471	85
	2		1.25	32		201617	84
	3		1.21	34		179566	86
	Mean	(+)	1.23	34	(+)	187551	85
2.00	1		1.41	24		326248	74
	2		1.44	22		359633	71
	3		1.49	19		415608	67
	Mean	(+)	1.45	22	(+)	367163	71
0.632	1		1.69	8		774327	38
	2		1.71	8		809588	35
	3		1.72	7		834314	33
	Mean	(+)*	1.71	8	(+)	806076	35
0.200	1		1.78	4		993012	20
	2		1.84	0		1217903	2
	3		1.86	0		1262208	-1
	Mean	(-)	1.83	1	(-)	1157708	7
0.0632	1		1.76	5		953037	24
	2		1.75	5		924992	26
	3		1.80	2		1074839	14
	Mean	(-)	1.77	4	(-)	984289	21
Control	1		1.93			1576718
	2		1.82			1118903
	3		1.88			1373426
	4		1.78			989789
	5		1.85			1222040
	6		1.84			1198036
	Mean		1.85			1246485

 

* = The concentration level 0.632 is considered to be not biologically significant, due to an inhibition ≤ 8%

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

In this study, the substance was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 9.30 (7.77 – 11.3) and 1.11 (0.816 – 1.47) mg/L. The NOEC-values for inhibition of growth rate and yield after 72 hours were 0.632 and 0.200 mg/L.

Executive summary:

Summary

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) and Council Regulation (EC) No.761/2009/Method C.3 (2009) at the test facility from 2016 -02 -08 to 2016 -02 -12, with the definitive exposure phase from 2016 -02 -09 to 2016 -02 -12. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4847 cells/mL. Based on a preliminary test, 6 concentration levels were tested in a geometrical series with a dilution factor of √10 (nominal): 0.0632 - 0.200 - 0.632 - 2.00 - 6.32 - 20.0 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

All test concentrations of the test item and the control were analytically verified by HPLC-DAD analysis at the start of the exposure (0 hours) and the end of the exposure (72 hours). The measured concentrations of Hostavin PR-25 P at the start of the exposure were in the range of 92 % to 102 % of the nominal values. At the end of the exposure, they were in the range of 84 to 117 %.  All effect values given are based on the nominal test item concentrations.

NOEC, LOEC, EC - values of the test item (0-72 hours) based on the nominal test item concentration [mg/L].

                               

	Growth Rate Inhibition
NOEC	0.632
LOEC	2.00
ErC10	0.963 (0.506 – 1.67)
ErC20	2.72 (1.93 – 3.49)
ErC50	9.30 (7.77 – 11.3)
	Inhibition of Yield
NOEC	0.200
LOEC	0.632
EyC10	< 0.0632
EyC20	0.298 (< 0.0632 – 0.541)
EyC50	1.11 (0.816 – 1.47)

 

Description of key information

72h-EC50 (Desmodesmus subspicatus, growth rate) = 9.30 mg/L, 72h-EC10 (Desmodesmus subspicatus, growth rate) = 0.963 mg/L (OECD 201, 2017)

Key value for chemical safety assessment

EC50 for freshwater algae:

9.3 mg/L

EC10 or NOEC for freshwater algae:

0.963 mg/L

Additional information

In a reliable key study, toxicity of the submission substance to the green alga was determined according to OECD 201 (2011) and in compliance with GLP (RL1). The study was conducted with five test concentrations (nominal 0.0632, 0.200, 0.632, 2.00,  6.32 and 20.0 mg/L) under static conditions during 72 hours. Controls were run in parallel. All test concentrations and controls were analytically verified by HPLC-DAD analysis. Derived effect concentrations are based on the nominal test concentrations. Adverse effects on growth rate and yield of biomass followed a dose-response relationship. Derived effect concentrations on growth rate (r) and yield of biomass (y) were (nominal): 72h-ErC50 = 9.30 mg/L, 72h-ErC10 = 0.963 mg/L (OECD 201, 2017), 72h-NOErC = 0.632 mg/L; 72h-EyC50 = 1.11 mg/L, 72h-NOEyC = 0.200 mg/L (EC10 was not derived for yield of biomass). The study was considered reliable and adequate for the environmental hazard assessment for aquatic organisms.