6-[C12-18-alkyl-(branched, unsaturated)-2,5-dioxopyrrolidin-1-yl]hexanoic acid

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:

2013-01-28 to 2013-02-01

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:

6-[3-(C12-C18)-alkenyl(branched, unsaturated)-2,5-dioxopyrrolidin-1-yl]hexanoic acid

EC Number:

701-162-1

Molecular formula:

C22H37NO4 - C28H49NO4

IUPAC Name:

6-[3-(C12-C18)-alkenyl(branched, unsaturated)-2,5-dioxopyrrolidin-1-yl]hexanoic acid

Details on test material:

- Name of test material (as cited in study report): (Pentapropylensuccinimido)-capronic acid
- Physical state: Liquid, brown viscous
- Analytical purity: 95.0 % (w/w) (100 % minus by-products)
- Purity test date: 2011-11-15
- Lot/batch No.: ESD0009272
- Expiration date of the lot/batch: 2014-04-26
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 7.50 - 15.0 - 30.0 - 60.0 - 120 mg/L
- Sampling method: Each test item concentration and the control were analytically verified at the beginning and at the end of the test. Analytical evaluation of (Pentapropylensuccinimido)-capronic acid was carried out via LC-MS/MS. Samples at test start were taken from additional replicate without algae. At end of the test the samples were taken directly from the pooled test replicates.
- Sample storage conditions before analysis: All original samples were stored at room temperature until sample preparation. Prepared samples were stored at room temperature until 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 agitation.
- Eluate: Dilution water
- Differential loading: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

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:

0.24 mmol Ca+Mg/l

Test temperature:

min.: 21.0 max.: 22.5, mean value: 21.75 °C

pH:

Geometric mean measured
test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
82.2 7.75 8.14
41.3 7.85 8.20
10.4 7.94 8.26
9.26 7.95 8.34
2.17 7.99 8.41
Control 8.05 8.47

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 653967 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  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
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 59 98
20.0 2 26
10.00 -1 -3
1.00 0 1
0.10 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.:

38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 33.5-42.6 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 8.4-10.9 mg/L

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 was determined by calculation of statistical significance of growth rate and yield. As 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 a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.
Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. These findings were marked “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are significant.

Any other information on results incl. tables

Cell Densities

	Geometric mean measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
82.2		1		4332		8304	10150	17971
		2		4332		8769	10263	19736
		3		4332		7747	12078	22632
		Mean		4332		8273	10830	20113
41.3		1		4332		8593	23161	48110
		2		4332		6549	20377	47289
		3		4332		8929	21009	46493
		Mean		4332		8024	21516	47297
10.4		1		4332		13380	53142	263766
		2		4332		14628	68729	351574
		3		4332		14241	63970	338585
		Mean		4332		14083	61947	317975
9.26		1		4332		16707	73418	452705
		2		4332		14993	79085	459073
		3		4332		16597	78664	512821
		Mean		4332		16099	77056	474866
2.17		1		4332		19245	139035	769252
		2		4332		20654	137393	805262
		3		4332		21944	142221	887001
		Mean		4332		20614	139550	820505
Control		1		4332		22072	120520	670027
		2		4332		18789	106953	667480
		3		4332		19339	112529	615556
		4		4332		17333	116224	648295
		5		4332		19198	118810	700314
		6		4332		23758	112642	622129
		Mean		4332		20082	114613	653967

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

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

Geometric mean measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
82.2	1		0.474	71.6		13639	97.9
	2		0.505	69.8		15404	97.6
	3		0.551	67.0		18300	97.2
	Mean	(+)	0.510	69.5	(+)	15781	97.6
41.3	1		0.802	52.0		43778	93.3
	2		0.797	52.4		42957	93.4
	3		0.791	52.7		42161	93.5
	Mean	(+)	0.797	52.4	(+)	42965	93.4
10.4	1		1.37	18.1		259434	60.1
	2		1.47	12.4		347242	46.6
	3		1.45	13.1		334253	48.6
	Mean	(+)	1.43	14.5	(+)	313643	51.7
9.26	1		1.55	7.31		448373	31.0
	2		1.55	7.03		454741	30.0
	3		1.59	4.83		508489	21.7
	Mean	(+)^1)	1.57	6.39	(+)	470534	27.6
2.17	1		1.73	-3.26		764920	-17.8
	2		1.74	-4.17		800930	-23.3
	3		1.77	-6.10		882669	-35.9
	Mean	(+)^2)	1.75	-4.51	(+)^2)	816173	-25.6
Control	1		1.68			665695
	2		1.68			663148
	3		1.65			611224
	4		1.67			643963
	5		1.70			695982
	6		1.66			617797
	Mean		1.67			649635

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	1.63	1.70	1.72	1.68	0.0461	2.74	8.99
	2	1.47	1.74	1.83	1.68	0.189	11.3
	3	1.50	1.76	1.67	1.65	0.139	8.39
	4	1.39	1.90	1.72	1.67	0.262	15.7
	5	1.49	1.82	1.77	1.70	0.180	10.6
	6	1.70	1.56	1.71	1.66	0.0862	5.20
				Mean	1.67
				SD ±	0.02
				CV [%]	0.97

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study (Pentapropylensuccinimido)-capronic acid was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values (geometric mean measured test item concentrations). The NOEC-values for inhibition of growth rate and yield after 72 hours were 9.26 and 2.17 mg/L, respectively. The LOEC-values for inhibition of growth rate and yield after 72 hours were 10.4 and 9.26 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % confidence intervals after 72 hours were 38.0 (33.5 – 42.6) and 10.3 (10.0 – 10.8) mg/L, respectively.
All effect levels are given based on geometric mean measured concentrations of (Pentapropylensuccinimido)-capronic acid.

Executive summary:

The toxicity of (Pentapropylensuccinimido)-capronic acid (batch number: ESD0009272)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from 2013-01-28 to 2013-02-01, with the definitive exposure phase from 2013-01-29 to 2013-02-01. 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 levels were tested in a geometrical series with a dilution factor of 2 (nominal): 7.50 - 15.0 - 30.0 - 60.0 - 120 mg/L, corresponding to geometric mean measured test item concentrations of 2.17 - 9.26 - 10.4 - 41.3 - 82.2 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.

Various test concentrations of (Pentapropylensuccinimido)-capronic acid and the control were analytically verified by LC-MS/MS at test start and test end.

The measured concentrations of (Pentapropylensuccinimido)-capronic acid at test start were in the range of 28 to 77 % of the nominal values. After 72 hours the measured concentrations of (Pentapropylensuccinimido)-capronic acid were in the range of 30 to 66 %. Therefore, all effect values given are based on the geometric mean measured concentrations of the test item (Pentapropylensuccinimido)-capronic acid.

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

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

	Growth Rate Inhibition
NOEC	9.26
LOEC	10.4
ErC10	9.59 (8.42 – 10.9)
ErC20	14.6 (13.2 – 16.3)
ErC50	38.0 (33.5 – 42.6)
	Yield Inhibition
NOEC	2.17
LOEC	9.26
EyC10	8.46 (7.74 – 8.90)
EyC20	8.92 (8.34 – 9.25)
EyC50	10.3 (10.0 – 10.8)