Dimethyl 2,5-dioxocyclohexane-1,4-dicarboxylate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to current OECD-Guidelines and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl 2,5-dioxocyclohexane-1,4-dicarboxylate

EC Number:

228-528-9

EC Name:

Dimethyl 2,5-dioxocyclohexane-1,4-dicarboxylate

Cas Number:

6289-46-9

Molecular formula:

C10H12O6

IUPAC Name:

1,4-dimethyl 2,5-dioxocyclohexane-1,4-dicarboxylate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 7.68 - 19.2 - 48.0 - 120 - 300 mg/L
- Sampling method: Separate replicates for test item analysis after 0 h were prepared without algae. Samples at end of the test were taken directly from the test replicates of each test concentration.
- Sample storage conditions before analysis: All samples were stored at 6 +/- 2 °C if necessary

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 300 mg/L was freshly prepared
- Eluate: Dilution water
- Differential loading: 7.68 - 19.2 - 48.0 - 120 - 300 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No undissolved material

Test organisms

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): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22 °C, Min: 21 °C, Max: 23 °C

pH:

Test item concentration pH-value
Nominal Geometric mean measured
[mg/L] Start; 0 h End; 72 h
300 137 7.51 7.88
120 48.0 7.72 7.96
48.0 13.4 7.75 8.17
19.2 1.63 7.89 8.46
7.68 0.414 7.92 8.71
Control 7.95 9.04

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Concentrations and Recovery Rates of Succinmethylester
Sampling date 2009-06-15
Test start (0 h) 2009-06-18
Test end (72 h)
Start of analysis 2009-06-15 2009-06-18
Nominal concentration Succinmethylester
Geometric mean measured concentration
Test item

[mg/L] Main component
[mg/L] Measured
Concentration *
[mg/L] Measured
Concentration *
[mg/L] RR

[%] Main component
[mg/L] Test item

[mg/L]
300 276 132 121 92 126 137
120 110 50.9 38.3 75 44.2 48.0
48.0 44.2 23.7 6.36 27 12.3 13.4
19.2 17.7 8.14 0.275 3 1.50 1.63
7.68 7.07 3.23 < LOQ 0.3811) 0.414
Control < LOQ < LOQ < LOQ

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- 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: 4878 cells/mL
- Control end cells density: Mean 804533 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


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



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: 1 - 10 - 100 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

51.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 39.1-67.6

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):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.695 (CI 0.657 - 0.729) mg/L
Yield Inhibition: 0.32 (CI 0.294 - 0.358) mg/L

Reported statistics and error estimates:

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

Any other information on results incl. tables

NOEC, LOEC, EC-Values and 95 % Confidence Intervals of Succinmethylester
(0-72 h)

based on geometric mean measured concentrations of the test item [mg/L]

                               

	Rate-related inhibition
NOEC	1.63
LOEC	13.4
ErC10	9.06 (6.16 – 12.2)
ErC20	15.2 (11.6 – 18.8)
ErC50	51.3 (39.1 – 67.6)
	Yield inhibition
NOEC	1.63
LOEC	13.4
EyC10	3.30 (2.20 – 4.85)
EyC20	4.68 (3.39 – 6.64)
EyC50	10.5 (7.53 – 14.3)

Cell Densities

	Test item concentration				Replicate		Cell density [cells/mL]
	Nominal		Geometric mean measured
[mg/L]				No.		0 h		24 h	48 h	72 h
300		137		1		4878		31216*	41207*	33944*
				2		4878		23259*	40346*	36039*
				3		4878		23392*	38980*	31558*
				Mean		4878		25956*	40178*	33847*
120		48.0		1		4878		22554*	54886*	44328*
				2		4878		23690*	63843*	81864*
				3		4878		35360*	80548*	81041*
				Mean		4878		27201*	66426*	69078*
48.0		13.4		1		4878		20796*	90629*	253489*
				2		4878		28690*	133802*	419943*
				3		4878		34045*	117772*	346922*
				Mean		4878		27844*	114068*	340118*
19.2		1.63		1		4878		25661	168647	706686
				2		4878		24888	180002	747788
				3		4878		35458	209823	1005368
				Mean		4878		28669	186157	819947
7.68		0.414		1		4878		23773	192941	800236
				2		4878		26463	222839	909319
				3		4878		30460	203749	964171
				Mean		4878		26899	206510	891242
Control				1		4878		17979	133858	621679
				2		4878		21135	171394	802104
				3		4878		26077	164910	766019
				4		4878		27192	213225	1003016
				5		4878		26655	167057	809237
				6		4878		22256	154396	825143
				Mean		4878		23549	167473	804533

Evaluation after 72 h

                        Statistically significant differences of growth rates and yield compared to

                                control values are marked (+), not significant differences are marked (-).

Test item concentration		Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
Nominal	Geometric mean measured
[mg/L]		[mg/L]	[d^-1]		[%]	[cells/mL]		[%]
300	137	1		0.647	61.93		29066	96.37
		2		0.667	60.75		31161	96.10
		3		0.622	63.36		26680	96.66
		Mean	(+)	0.645	62.01	(+)	28969	96.38
120	48.0	1		0.736	56.69		39450	95.07
		2		0.940	44.65		76986	90.37
		3		0.937	44.85		76163	90.48
		Mean	(+)	0.871	48.73	(+)	64200	91.97
48.0	13.4	1		1.317	22.47		248611	68.91
		2		1.485	12.57		415065	48.09
		3		1.421	16.32		342044	57.23
		Mean	(+)	1.408	17.12	(+)	335240	58.08
19.2	1.63	1		1.659	2.35		701808	12.24
		2		1.677	1.24		742910	7.10
		3		1.776	-4.56		1000490	-25.12
		Mean	(-)	1.704	-0.32	(-)	815069	-1.93
7.68	0.414	1		1.700	-0.09		795358	0.54
		2		1.743	-2.59		904441	-13.10
		3		1.762	-3.74		959293	-19.96
		Mean	(-)	1.735	-2.14	(-)	886364	-10.84
Control		1		1.616			616801
		2		1.701			797226
		3		1.685			761141
		4		1.775			998138
		5		1.704			804359
		6		1.710			820265
		Mean		1.699			799655

Section-by-Section and Average Specific Growth Rates of the Control Group

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 – 24 h	24 – 48 h	48 – 72 h
Control	1	1.304	2.008	1.536	1.616	0.3584	22.2	14.3
	2	1.466	2.093	1.543	1.701	0.3418	20.1
	3	1.676	1.844	1.536	1.685	0.1545	9.16
	4	1.718	2.059	1.548	1.775	0.2603	14.7
	5	1.698	1.835	1.578	1.704	0.1289	7.56
	6	1.518	1.937	1.676	1.710	0.2116	12.4
				Mean	1.699
				SD ±	0.05
				CV [%]	3.01

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Succinmethylester was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (geometric mean measured test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 51.3 (39.1 – 67.6) and 10.5 (7.53 – 14.3) mg/L, respectively. All effect levels are given based on geometric mean measured concentrations of Succinmethylester.

Executive summary:

The toxicity of Succinmethylester(Batch no.: DEB2 075992) to the unicellular freshwater green alga Desmodesmus subspicatuswas determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 , from June 15 to 23, 2009. The aim of the study was to assess the effects on growth rate and yield production over a period of 72 h.
The study was conducted under static conditions with an initial cell density of approximately 2 x 5 x 10³cells/mL. Five concentration levels were tested in a geometrical series with a dilution factor of √10, nominal: 7.68 - 19.2 - 48.0 -120 - 300 mg/L, corresponding to geometric mean measured test item concentrations of 0.414 - 1.63 - 13.4 - 48.0 - 137 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.

The concentrations of Succinmethylester were analysed at test start and end via HPLC analysis. The recovery rates after 72 h related to initial measured concentrations show a concentration related decrease from 92 % to LOQ. Therefore all effect levels are given based on geometric mean measured concentrations of the test item

NOEC, LOEC, EC-Values and 95 % Confidence Intervals of Succinmethylester
(0-72 h)

                                based on geometric mean measured concentrations of the test item [mg/L]

                               

	Rate-related inhibition
NOEC	1.63
LOEC	13.4
ErC10	9.06 (6.16 – 12.2)
ErC20	15.2 (11.6 – 18.8)
ErC50	51.3 (39.1 – 67.6)
	Yield inhibition
NOEC	1.63
LOEC	13.4
EyC10	3.30 (2.20 – 4.85)
EyC20	4.68 (3.39 – 6.64)
EyC50	10.5 (7.53 – 14.3)