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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation 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:

no

Details on sampling:

- Concentrations: 2.50 - 5.00 - 10.0 - 20.0 - 40.0 - 80.0 mg/L (dilution factor 2).
- Sampling method: All test concentrations and the control were analytically verified by LC-MS/MS at the start of the exposure (0 h) and at the end of the exposure (48 h) as specified below.
Sampling at the start of the exposure (0 hours) was carried out immediately before application.
At the end of the exposure (48 hours), samples were taken directly from the test vessels, which contained the daphnids.
- 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 was freshly prepared in demineralised water, which was adjusted with 1 M NaOH to a pH of 11 ± 0.2 (actual value was pH 10.8).
80.0 mg of the test item were weighed out and transferred with 900 mL of the demineralised water (pH 10.8).
The stock solution was stirred with approximately 1100 rpm for 24 hours at room temperature.
After the stirring period, the mineral components as specified under 'Dilution water' were added to yield the highest test concentration.
- Eluate: Dilution water
- Differential loading: 2.50 - 5.00 - 10.0 - 20.0 - 40.0 - 80.0 mg/L (dilution factor 2).
- Controls: Dilution water without test item tested under the same conditions as the test groups.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In 2-3 L glass vessels with appr. 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µExm-2 x s-1
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

DILUTION WATER:
Dilution water ISO test water, according to OECD 202, Annex 3:
Component Concentration [mg/L]
KCl 5.76
NaHCO3 64.8
CaCl2  2 H2O 294
MgSO4  7 H2O 123
pH-value: 7.8  0.2

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water at test start: 174 (mg CaCO3/L)

Test temperature:

20.4 °C (Water)

pH:

Water Quality Parameters at the Start of the Exposure (0 h)
(measured in one additional replicate per concentration level and control)
Nominal concentration
of the test item
[mg/L] pH-Value


80.0 8.96
40.0 8.74
20.0 7.99
10.0 7.76
5.00 7.69
2.50 7.81
Control 7.88

Water Quality Parameters at the End of the Exposure (48 h)
(measured in all replicates)
Nominal concentration
of the test item
[mg/L] pH-Values
Replicates
1 2 3 4
80.0 7.57 7.59 7.55 7.56
40.0 7.56 7.55 7.53 7.54
20.0 7.56 7.55 7.54 7.54
10.0 7.53 7.54 7.56 7.56
5.00 7.54 7.56 7.57 7.56
2.50 7.54 7.55 7.55 7.63
Control 7.56 7.59 7.60 7.59

Dissolved oxygen:

Water Quality Parameters at the Start of the Exposure (0 h)
(measured in one additional replicate per concentration level and control)
Water Quality Parameters at the Start of the Exposure (0 h)
(measured in one additional replicate per concentration level and control)
Nominal concentration
of the test item
[mg/L] Dissolved O2-Concentration

[mg/L]
80.0 8.30
40.0 8.36
20.0 8.44
10.0 8.44
5.00 8.46
2.50 8.44
Control 8.38


Water Quality Parameters at the End of the Exposure (48 h)
(measured in all replicates)
Nominal concentration
of the test item
[mg/L] Dissolved O2 -Concentration [mg/L]
Replicates
1 2 3 4
80.0 6.73 6.84 6.68 7.02
40.0 6.92 6.97 7.03 7.09
20.0 7.00 7.29 7.07 7.75
10.0 7.16 7.29 7.75 5.63
5.00 7.16 7.45 7.35 7.40
2.50 7.15 7.85 6.90 7.22
Control 7.64 8.00 8.08 7.20

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: see above
- Conductivity: Dilution water at test start:632 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility was determined in all groups after 24 h and 48 h.

TEST CONCENTRATIONS
- Range finding study:
Immobilisation Rates in the non GLP Preliminary Range Finding Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item
Concentration
[mg/L] IMMOBILISATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
100 100 100 100 100 100 100
10.0 10 0 5 30 10 20
1.00 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI 1.9-3.0 mg/L

Details on results:

- Mortality of control: 0 %
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: 1.69 (CI 1.41 - 2.12) mg/L

Reported statistics and error estimates:

EC10- and EC50-values were calculated by sigmoidal dose-response regression. The confidence intervals were calculated from the best-fit values, the standard error and the
t-distribution with the software GraphPad prism5. The concentration effect relationships are presented graphically.

Any other information on results incl. tables

Immobilisation Rates in the Definitive Test after 24 and 48 h of Exposure

                 (n = 20, divided into 4 replicates with 5 daphnids each)

(Pentapropylensuccinimido)-capronic acid		IMMOBILISATION [%]
Nominal Test Item Concentration [mg/L]	Geometric Mean Measured Test Item Concentration [mg/L]	24 h					48 h
		Replicates					Replicates
		1	2	3	4	MV	1	2	3	4	MV
80.0	56.8	100	100	100	100	100	100	100	100	100	100
40.0	25.1	100	100	100	100	100	100	100	100	100	100
20.0	12.6	60	40	20	0	30	100	100	100	100	100
10.0	6.23	40	0	0	0	10	60	80	80	60	70
5.00	3.13	20	20	60	40	35	80	80	80	100	85
2.50	1.57	0	0	0	0	0	0	0	0	0	0
Control		0	0	0	0	0	0	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the geometric mean measured concentrations of the test item (Pentapropylensuccinimido)-capronic acid, the 48-h EC50 for Daphnia magna was estimated to be 2.59 mg/L (95 % confidence limits: 1.85 – 2.97 mg/L).
The NOEC after 48 h was 1.57 mg/L. The LOEC after 48 h was 3.13 mg/L.

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the test item (Pentapropylensuccinimido)-capronic acid (batch number: ESD0009272) were determined according to OECD 202 (2004) from 2013-01-28 to 2013-02-01, with the definitive exposure phase from 2013-01-29 to 2013-01-31, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

The study was conducted under static conditions over a period of 48 h. Six concentration levels of the test item (Pentapropylensuccinimido)-capronic acid in the range of 2.50 to 80.0 mg/L were prepared with dilution water in a geometric series with a separation factor of 2 and tested. Twenty daphnids were exposed to each concentration level and the control.

The concentrations of the test item (Pentapropylensuccinimido)-capronic acid were analytically verified by LC-MS at the start of the exposure (0 h) and at the end of the exposure (48 h) of all concentration levels and the control. Details of the analytical method are presented in part 11.

The measured concentrations of the test item at the start of the exposure (0 h) were in the range of 83 to 102 % of the nominal values. The measured concentrations of the test item at the end of the exposure (48 h) were in the range of 44 to 49 % of the nominal values. The geometric mean measured concentrations were calculated to be: 1.57 – 3.13 – 6.23 – 12.6 – 25.1 – 56.8 mg/L. The analytical results are presented in Table 4. The endpoints were assessed based on the geometric mean measured concentrations of the test item (Pentapropylensuccinimido)-capronic acid (Table 1).

The water quality parameters (i.e. pH-values and dissolved oxygen concentrations), measured at the start (0 h) and the end of the exposure (48 h), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

EC₁₀-, EC₅₀- (with Confidence Interval), EC₁₀₀-Values

                   (based on the geometric mean measured concentrations of the test item)

Endpoints	Test Duration [h]	(Pentapropylensuccinimido)-capronic acid
		Geometric Mean Measured Concentrations of the Test Item [mg/L]	95 % confidence limits [mg/L]
EC10	24	10.4	7.31 – 18.6
	48	2.04	1.57 – 2.80
EC50	24	14.2	12.4 – 21.6
	48	2.59	1.85 – 2.97
EC100	24	25.1	Not applicable
	48	12.6	Not applicable
NOEC	48	1.57	Not applicable
LOEC	48	3.13	Not applicable