N-methyl-N-[C18-(unsaturated)alkanoyl]glycine

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Nov - 06 Dec 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

02 Oct 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD guidance no. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures

Version / remarks:

14 Dec 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany (08 Apr 2015)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples for analytical determinations were taken from the new solutions at the beginning of the tests, and from the old solutions (with and without daphnia and algae) at the end of the test (set 1).
Additional samples from the new and the old solutions were taken on day 5, day 7, day 9 and day 12. In the control, the content of the test item was measured at the beginning and at the end of each study.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For the test, a saturated solution of 100 mg/L was prepared by mixing respective amounts of test item in dilution water and shaking vigorously for 24 h. The resulting solution was then filtered through a 0.45 µm cellulose ester filter after which the solutions were clear and colorless. The lower concentrations for both Experiments were prepared by dilution of the membrane filtered, saturated solution with dilution water.
- Differential loading: No
- Controls: Dilution water without test item
- Evidence of undissolved material: No, the solutions were clear and colorless after filtration.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Planktonic crustacean / water flea
- Strain/clone: Berlin
- Source: Umweltbundesamt Berlin, Germany (27 Sep 2007)
- Animal husbandry: In-house according to ES IS 6341, following SOP 115 002 01, edition 12 (adopted 12 Feb 2015). Vessels: 2L glass beakers; Medium: M4 medium (Elendt); Food: Green alga Desmodesmus subspicatus, twice a week; Photo period: 16 h light / 8 h dark (neon tubes); Temperature: 20 ± 2 °C
- Feeding during test: The animals were fed on each working day. On weekends, the Daphnia were not fed. On Fridays, the double amount of alga suspension was added into the test vessels.
- Sex and age at test start: Females between 0 - 24 h old

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
23 h before the start of set 1 and 21 h before the start of set 2, the adult animals were separated from the young. 22 h (set 1) and 20 h (set 2) later, the adults were caught with the help of a glass tube, and the newborn daphnia, aged 0 - 23 h (set 1) and 0 - 22 h (set 2), were sieved from the medium and immediately placed into a 250 mL beaker containing diliution water. After a settling period of 30 min, animals which showed no apparent damage were used for the tests.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

267 - 356 mg CaCO3/L

Test temperature:

19.5 - 21.5 °C

pH:

7.5 - 8.7

Dissolved oxygen:

8.5 - 9.7 mg/L

Nominal and measured concentrations:

Control, 4.6, 10, 22, 46 and 100 mg/L (nominal)
Control, 27, 47, 102, 183 and N.A. µg/L (time-weighted mean)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers
- Renewal rate of test solution: 3 times/week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 80 mL/animal, animals were kept individually
- Other: 1 additional replicate for analytical measurement

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water enriched with minerals, according to the M4 composition by Elendt, as described in the OECD guideline.
- CaCl2*2H2O: 293.80 mg/L
- MgSO4*7H2O: 123.30 mg/L
- NaHCO3: 64.80 mg/L
- KCl: 5.80 mg/L
- NaNO3: 274 µg/L
- K2HPO4: 143 µg/L
- Na2SiO3*9H2O: 10 mg/L
- H3BO3: 2.86 mg/L
- Na2EDTA*2H2O: 2.5 mg/L
- FeSO4*7H2O: 0.9955 mg/L
- MnCl2*4H2O: 0.3605 mg/L
- LiCl: 0.306 mg/L
- SrCl2*6H2O: 0.152 mg/L
- RbCl: 0.071 mg/L
- Na2MoO4*2H2O: 0.0615 mg/L
- CuCl2*2H2O: 16.75 µg/L
- NaBr: 16 µg/L
- ZnCl2: 13 µg/L
- CoCl2*6H2O: 10 µg/L
- KI: 3.25 µg/L
- Na2SeO3 *5 H2O: 3.33 µg/L
- NH4VO3: 0.575 µg/L
- ThiaminHCl: 75 µg/L
- Cyanocobalamin: 1 µg/L
- D+Biotin: 0.75 µg/L
- Culture medium different from test medium: Culture medium same as dilution water (M4-medium).
- Intervals of water quality measurement: Day 2, 5, 12 and 19

OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 1000 lux (neon tubes)

EFFECT PARAMETERS MEASURED:
- Mortality and Reproduction of surviving daphnia: 3 times/week

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2.17
- Range finding study: Not specified

Reference substance (positive control):

not specified

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 183 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

102 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Behavioural abnormalities: No abnormal behaviour or appearance of the surviving daphnia was observed.
- Mortality of control: 0%
- Any observations that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Reported statistics and error estimates:

Statistical analyses were performed with ToxRat Professional v3.2.1.
Statistical design: Hypothesis testing (NOEC) and regression (ECx).

VALIDITY CRITERIA

The study fulfilled the validity criteria defined by the guideline (Table 1). Furthermore, the oxygen concentration was ≥ 8.5 mg/L (required: ≥, 3.0 mg/L) the pH range 7.5 - 8.4 (required 6 - 9) and the pH variation was 0.6 (required: ≤ 1.5). Thus, the test is considered valid and reliable.

Table 1: Validity criteria for OECD 211.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals in the control (femaleDaphnia) does not exceed 20% at the end of the test.	Mortality of parent animals in the control was 0%.	Yes
The mean number of living offspring produced per parent animal surviving at the end of the test is ≥ 60 in the control.	The mean surviving offspring in the control was 93.	Yes

 

ANALYTICAL RESULTS

There was a weak correlation between the nominal concentrations and the measured concentrations and a weak stability of the test item during the exposure period (Table 2 - 5). In aged test solutions, hardly any test item was detectable. Therefore, results were based on the time-weighted mean of the measured concentrations in the new and old solutions. For the calculation of the time-weighted mean, the limit of detection (10 µg/L) was used for calculation where no test item was detectable.

Table 2.Measured Concentrations Days 0 – 5 set 1

Nominal Conc. in mg/L	Measured Conc. day 0 in µg/L	Measured Conc. day 5 old solution with algae in µg/L	Measured Conc. day 5 old solution without algae in µg/L	Measured Conc. day 5 new solution algae in µg/L
4.6	126	10**	n. d.	62
10	252	10**	n. d.	151
22	514	31	n. d.	344
46	1061	41	87	657
100	2177	*	*	*

n. d. = not detectable

* not measured because all daphnia were found dead on day 2

**10 µ/L = LOD (lowest calibration level)

Table 3.Measured Concentrations Days 7 – 9 set 1

Nominal Conc. in mg/L	Measured Conc. day 7 old solution with algae in µg/L	Measured Conc. day 7 old solution without algae in µg/L	Measured Conc. day 7 new solution algae in µg/L	Measured Conc. day 9 old solution with algae in µg/L	Measured Conc. day 9 old solution without algae in µg/L
4.6	10**	n. d.	40	10**	n. d.
10	10**	n. d.	100	10**	n. d.
22	12	n. d.	255	45	n. d.
46	37	38	417	92	11

n. d. = not detectable

**10 µ/L = LOD (lowest calibration level)

 

Table 4.Measured Concentrations Days 9 – 21 set 1

Nominal Conc. in mg/L	Measured Conc. day 9 new solution algae in µg/L	Measured Conc. day 12 old solution with algae in µg/L	Measured Conc. day 12 old solution without algae in µg/L	Measured Conc. day 12 new solution algae in µg/L	Measured Conc. day 21 old solution with algae in µg/L
4.6	53	10**	n. d.	30	10**
10	108	10**	n. d.	78	10**
22	271	10**	n. d.	172	17
46	481	15	n. d.	327	22

n. d. = not detectable

**10 µg/L = LOD (lowest calibration level)

Table 5. Time-Weighted Mean set 1

Nominal Conc. in mg/L	Time-Weighted Mean in µg/L
4.6 mg/L	27
10 mg/L	47
22 mg/L	102
46 mg/L	183

 

BIOLOGICAL RESULTS

No toxicity (mortality and inhibition) was observed at the nominal concentration of 22 mg/L (experimental set 1). Therefore, the lower concentrations of 1.0 and 2.2 mg/L, which were additionally tested in the experimental set 2, were not necessary for evaluation and the entire set 2 was aborted on Day 14.

At the nominal concentration of 46 mg/L only 1 animal survived but the number of offspring of the surviving animal was not significant compared to the control. Thus, no statistically significant inhibition of reproduction was observed in any treatment (Table 6). The effect values based on the time-weighted mean of the measured concentrations are summarized in Table 7 and Table 8.

Table 6. Fecundity (set 1)

Treatment	Total Offspring	Surviving Parent Animals	Mean Offspring per Surviving Parent Animal	Inhibition of Reproductionin %
Blank Control	934	10	93	0
4.6 mg/L	1030	10	103	-10
10 mg/L	952	10	95	-2
22 mg/L	1098	10	110	-18
46 mg/L	77	1	77	18
100 mg/L	0	0	--	--

 

Table 7.Biological Results cumulative offspring per survived parent

Parameter	Value
NOEC	≥183 µg/L
LOEC	> 183 µg/L
EC50	> 183 µg/L

 

Table 8.Biological Results Immobility

Parameter	Value
NOEC	102 µg/L
LOEC	183 µg/L
EC50	141.46 µg/L

 

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

NOEC (21d) ≥ 0.183 mg/L for Daphnia magna (meas. TWA, OECD 211)

Additional information

One study investigating the long-term toxicity of N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC 701-177-3) to aquatic invertebrates is available. The study was performed according to OECD 211 (GLP) with Daphnia magna under semi-static conditions with a renewal rate of test solution three times/week. The test organisms were exposed to nominal concentrations of 4.6, 10, 22, 46 and 100 mg/L. The content of the test item in the test solutions was determined by measurement of Oleoylsarcosine the main component of the test item using LC-MS-MS determination. The analysis of the test item concentrations in the treatments before and after exposition showed weak correlation between the nominal concentrations and the measured concentrations and weak stability of the test item during exposition. Therefore, the timeweighted mean of the measured concentrations in the new and the old solutions was calculated and used for the evaluation of the biological results (27, 47, 102, 183 and N.A. µg/L time-weighted mean). After only 2 days strong mortality was observed in the two highest concentrations (100% mortality at 100 mg/L nominal and 90% mortality at 46 mg/L nominal until the test end (day 21)). Therefore, parallel to the ongoing experiment (set 1), on day 7, additional control and test replicates using lower test concentrations of 1.0 mg/L and 2.2 mg/L were started (set 2). As no toxicity, neither mortality nor inhibition of reproduction was observed in the first experiment at the nominal concentration 22 mg/L, the additional tested lower concentrations 1.0 mg/L and 2.2 mg/L were considered not necessary for evaluation and set 2 was aborted on day 14. A statistically significant difference of the reproduction rate between control and a nominal test concentration of 46 mg/L was not determined. Thus, a NOEC (21d) of ≥ 183 µg/L (meas. TWA) based on the reproduction rate was reported although only one parental daphnia survived until the test end (day 21) at the test concentration of nominal 46 mg/L (183 µg/L meas. TWA).