Dihydro-3-(tripropenyl)furan-2,5-dione

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:

2013-02-19 to 2013-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study, no deficiencies

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

Tripropenyl succinic acid

Cas Number:

147384-84-7

Molecular formula:

R-C4H6O4 , whereas R=C8-C10-alkyl-(branched, unsaturated)

IUPAC Name:

Tripropenyl succinic acid

Details on test material:

- Name of test material (as cited in study report): Tripropenyl succinic acid
- Physical state: Viscous liquid, pale brown
- Analytical purity: 99.3 %
- Purity test date: 2013-03-04
- Lot/batch No.: 12-SK002
- Expiration date of the lot/batch: January 2014
- Stability under test conditions: Not applicable
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 2.56 - 6.40 - 16.0 - 40.0 - 100 mg/L
- Sampling method: All test item concentrations and the control were analytically verified via LC-MS/MS at the beginning and end of the test. Samples at test start were taken from an additional replicate without algae. At end of the test samples were taken directly from the pooled test replicates.
- Sample storage conditions before analysis:All samples were stored at room temperature before preparation and before analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was freshly prepared with dilution water. Dispersion treatment was 1 min ultrasound at room temperature
- Eluate: Dilution water
- Differential loading: 2.56 - 6.40 - 16.0 - 40.0 - 100 mg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test vessels.

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): 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.

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:

0.12 mmol Ca+Mg/l

Test temperature:

min.: 21.0 max.: 22.0, mean value: 21.5 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
100 6.76 6.93
40.0 8.10 8.28
16.0 8.17 8.33
6.40 8.22 8.37
2.56 8.25 8.42
Control 8.28 8.50

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 103 cells/mL, Actual: mean 4332 cells/mL
- Control end cells density: Mean 741639 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 with reduced concentration of magnesium and calcium
Composition of Dilution Water
Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 6*
CaCl2  2 H2O 9*
MgSO4  7 H2O 7.5*
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  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: 67.9



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.



TEST CONCENTRATIONS

Results of the Range Finding Test (0-72 hours)

Nominal Test Item Concentration
[mg/L] Growth Rate Inhibition[%] Yield Inhibition [%]
100 10 44
10.0 2 10
1.00 0 - 2

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

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

Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.631 0.682 ± 0.452
95 %
confidence
interval 0.583 - 0.687
Yield Inhibition
EyC50 0.256 0.314 ± 0.144
95 %
confidence
interval 0.247 - 0.265
SD = Standard deviation

Reported statistics and error estimates:

NOEC/LOEC were 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.

Any other information on results incl. tables

Cell Densities

Nominal test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
100	1	4332	18342	98814	552279
	2	4332	15798	100631	573759
	3	4332	16956	103990	533314
	Mean	4332	17032	101145	553117
40.0	1	4332	18883	131443	962355
	2	4332	16320	143712	888227
	3	4332	19726	144919	966884
	Mean	4332	18310	140025	939155
16.0	1	4332	20619	125958	845739
	2	4332	15575	134273	891593
	3	4332	20082	132556	796551
	Mean	4332	18759	130929	844628
6.40	1	4332	15348	133069	785826
	2	4332	14587	132075	781077
	3	4332	20824	129468	782650
	Mean	4332	16920	131537	783184
2.56	1	4332	16874	120068	672795
	2	4332	16424	117580	809477
	3	4332	18201	125467	707799
	Mean	4332	17166	121038	730024
Control	1	4332	16468	123313	702138
	2	4332	14182	132597	719530
	3	4332	16767	120942	749502
	4	4332	18097	113488	722612
	5	4332	19122	138114	782493
	6	4332	19503	118391	773561
	Mean	4332	17357	124474	741639

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), non 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]		[%]
100	1		1.62	5.72		547947	25.7
	2		1.63	4.98		569427	22.8
	3		1.60	6.40		528982	28.3
	Mean	(+)	1.62	5.70	(+)	548785	25.6
40.0	1		1.80	-5.08		958023	-29.9
	2		1.77	-3.52		883895	-19.9
	3		1.80	-5.17		962552	-30.6
	Mean	(+)*	1.79	-4.59	(+)*	934823	-26.8
16.0	1		1.76	-2.57		841407	-14.1
	2		1.78	-3.60		887261	-20.3
	3		1.74	-1.40		792219	-7.45
	Mean	(+)*	1.76	-2.52	(+)*	840296	-14.0
6.40	1		1.73	-1.14		781494	-5.99
	2		1.73	-1.02		776745	-5.35
	3		1.73	-1.06		778318	-5.56
	Mean	(-)	1.73	-1.07	(-)	778852	-5.63
2.56	1		1.68	1.88		668463	9.34
	2		1.74	-1.72		805145	-9.20
	3		1.70	0.89		703467	4.59
	Mean	(-)	1.71	0.35	(-)	725692	1.58
Control	1		1.70			697806
	2		1.70			715198
	3		1.72			745170
	4		1.71			718280
	5		1.73			778161
	6		1.73			769229
	Mean		1.71			737307

Section-by-Section and Average Specific Growth Rates of the Control Group (0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.34	2.01	1.74	1.70	0.341	20.1	18.2
	2	1.19	2.24	1.69	1.70	0.525	30.8
	3	1.35	1.98	1.82	1.72	0.325	18.9
	4	1.43	1.84	1.85	1.71	0.239	14.0
	5	1.48	1.98	1.73	1.73	0.246	14.2
	6	1.50	1.80	1.88	1.73	0.197	11.4
				Mean	1.71
				SD ±	0.01
				CV [%]	0.84

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Tripropenyl succinic acid was found to slightly inhibit the growth of the freshwater green alga Desmodesmus subspicatus over the period of 72 hours. The EC50-values for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 mg test item/L. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 40.0 mg test item/L.
All effect values given are based on nominal test item concentrations of Tripropenyl succinic acid.

Executive summary:

The toxicity of Tripropenyl succinic acid(batch number: 12-SK002)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from 2013-02-19 to 2013-02-22. 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 4332 cells/mL. Based on a preliminary test, 5 concentration were tested in a geometrical series with a dilution factor of 2.5, nominal: 2.56 - 6.40 - 16.0 - 40.0 - 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were within the acceptable limits.

The concentrations of Tripropenyl succinic acid were analytically verified by LC-MS/MS at test start and test end of all test concentrations and the control.

The measured concentrations of Tripropenyl succinic acid at test start were in the range of 111 to 120 % and after 72 hours in the range of 112 to 119 % of the nominal values. Therefore, all effect values given are based on the nominal concentrations of the test item Tripropenyl succinic acid

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Tripropenyl succinic acid
(0-72 hours)

                   based on nominal test item concentrations [mg/L]

	Inhibition of Growth Rate
NOEC	40.0
LOEC	100
ErC10	> 100
ErC20	> 100
ErC50	> 100
	Inhibition of Yield
NOEC	40.0
LOEC	100
EyC10	76.2 (69.3 – 84.9)
EyC20	90.8 (81.7 – > 100)
EyC50	> 100