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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Aug - 03 Sep 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 1% (w/v) solution with acetone was prepared. After evaporation of the solvent dilution water was added.
- Chemical name of vehicle: acetone

Test organisms (species):

Leuciscus idus

Details on test organisms:

TEST ORGANISM
- Common name: Golden orfe
- Source: Obtained from fish farm Paul Eggers, Hohenweststedt, Germany on 07 Jul 1982
- Length at study initiation: 5.9 cm (range 5.6 - 6.5 cm)
- Weight at study initiation: 1.9 g (range 1.4 - 2.7 g)

CULTURE
- Conditions: Fish were held in dechlorinated tap water that was passed through charcoal, under continuous aeration and flow-through conditions
- Health during culture (any mortality observed): No mortality during 2 weeks prior to test start
- Medical treatment: Once with 0.05 mg/L malachite green chloride, once with 10 mg/L tetracyclin hydrochloride

ACCLIMATION
- Acclimation period: 3 days
- Acclimation conditions: Same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

20 - 22 °C

pH:

7.7 - 7.9

Dissolved oxygen:

8.2 - 9.1 mg/L

Nominal and measured concentrations:

Nominal: 1.47, 2.15, 3.16, 4.64, 6.81, 10.0, 14.7, 21.5, 31.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquaria
- Material, size, fill volume: Glass, 30 x 22 x 24 cm, 10 L
- Aeration: Slight aeration
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- Biomass loading rate: 1.9 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised tap water was used for the preparation of reconstituted water (equivalent to recommendations in OECD 203)
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: Temperature, pH and oxygen concentration were recorded at test start and every 24 h

OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Mortality and behavioural abnormalities were recorded after 1, 4, 24, 48, 72 and 96 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 1.5

Reference substance (positive control):

yes

Remarks:

chloroacetamide

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

6.81 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Fish exhibited apathy after 48 h at 10.0, 14.7 and 21.5 mg/L and after 4 h at 31.6 mg/L.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Particles of undissolved test substance were visible at the bottom of the test aquaria. Concentration increase involved an increase of particles.

Results with reference substance (positive control):

LC50 (96h)= approx. 38 mg/L

Sublethal observations / clinical signs:

As for the test concentrations 10.0, 14.7, 21.5 and 31.6 mg/L effects were observed, it could not be excluded that these are physical effects due to the undissolved test material. Therefore, these test cocnentrations could be not evalueted as reliable. The test concentration 6.81 mg/L is the highest test concentration with no effects although undissolved test material was observed. Thus, this concentration and all concentrations below could be evaluated as reliable. It could be concluded that no effects were expected up to the water solubilty of the substance.

Table 1: Mortality of Leuciscus idus during exposure

Nominal Concentration (mg/L)	Number of Fish	% Mortality after 96 h
1.47	10	0
2.15	10	0
3.16	10	0
4.64	10	0
6.81	10	0
10.0	10	70
14.7	10	90
21.5	10	90
31.6	10	100

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Jul - 03 Aug 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted in 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

adopted in 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: At test start, at test end and every 24 h, the test item concentration was determined.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: On the day of the start of the study, a stock solution containing 10 g/L test item in deionised water was prepared. 10 g test item was mixed with 1.1 g NaOH (45%). Then, 40 mL deionised water (80 – 100 °C) was added. After homogenisation with a glass stirrer, 85 mL deionised water (room temperature) was added. Then the vessel was stirred on a magnetic stirrer. NaOH (0.5 M) was added until the resulting solution was clear. The pH of the solution was adjusted to 7.8 ± 0.2 with 1 M HCl. The solution was transferred into a volumetric flask and filled up to 1 L with deionised water. From this stock solution, a solution (nominal concentration: 25 mg/L) in drinking water was prepared. This solution then was used to prepare the solution of the concentration to be tested.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Strain: Hamilton-Buchanan
- Source: Received on 08 Jul 2010 from Dehner Garten Center, Neustadt/Weinstrasse, Germany
- Age at study initiation: Sexually immature young fish
- Length at study initiation: 2 ± 1 cm
- Feeding during test: None

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Warmwater fish food and daphnia, totalling to about 1 - 2% of body weight per day
- Feeding frequency: three times a day until 24h prior to test start
- Health during acclimation (any mortality observed): Mortality was < 5%.

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

1.18 mM

Test temperature:

21.7 - 22.3 °C

pH:

8.1 - 8.4

Dissolved oxygen:

7.7 - 9.1 mg/L

Nominal and measured concentrations:

Nominal: 0.43 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquaria
- Material, size: Glass, 10 L
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): approx. every 5 h
- No. of organisms per vessel: 8 (test item), 7 (control)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Drinking water
- Total organic carbon: < 1.0 mg/L
- Metals: Na 3.8 mg/L, K 2.4 mg/L, Ca 23.8 mg/L, Mg 1.9 mg/L, other metals < 0.0001 to < 0.01
- Pesticides: < LOD
- Chloride: 7.0 mg/L
- Ca/Mg ratio: 12.5:1
- Conductivity: 142 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: Oxygen concentration and pH were measured every 24 h.

OTHER TEST CONDITIONS
- Photoperiod: 12h light/12 h dark, neon tubes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Mortalities and abnormal behaviour were documented every 24 h.

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: Threshold approach concentration, lowest EC50 in alga and daphnia test

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Details on results:

- Behavioural abnormalities: None
- Mortality of control: None
- Mortality in the test vessels: None
- Analytics: 91% to 107% of the nominal concentration was measured at test start and every 24 h until test end.

Sublethal observations / clinical signs:

The substance showed a strong absorption, the test was performed with the highest achievable concentration in water.

ANALYTICAL RESULTS

The measured concentrations showed a very good correlation with the nominal concentrations (91 – 107%). Therefore, the nominal concentration was used for the determination of the results. The measured concentrations for treatment and control are given in Table 1.

Table 1. Analytical determinations

Nominal concentration	0 h		24 h		48 h
	Measured conc.	% of nom. conc.	Measured Conc.	% of nom. conc.	Measured conc.	% of nom. conc.
mg/L	mg/L	%	mg/L	%	mg/L	%
0	n.d.	-	n.d.	-	n.d.	-
0.43	0.46	107%	0.39	91%	0.44	102%
Nominal concentration	72 h		96 h
	Measured conc.	% of nom. conc.	Measured Conc.	% of nom. conc.
mg/L	mg/L	%	mg/L	%
0	n.d.	-	n.d.	-
0.43	0.42	97%	0.45	104%

n.d. = not detectable

Validity criteria fulfilled:

yes

Description of key information

LC50 (96h) > 0.43 mg/L for D. rerio (OECD 203)

Additional information

Two studies investigating the short-term toxicity of N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC 701-177-3) to freshwater fish are available.

The key study was carried out according to OECD guideline 203 and GLP. In this limit-test, zebra fish (Danio rerio) were exposed to 0.43 mg/L under flow-through conditions. Analytical measurements verified the test item concentration to be 91 – 107% throughout the exposure period. As no mortalities were observed, the LC50 was determined to be > 0.43 mg/L (nominal). Thus, no effects up to the water solubility were observed.

The supporting study was carried out equivalent to EU Method C.1 under non-GLP conditions without analytics. The test was conducted under static conditions with Golden orfe (Leuciscus idus) as test organism. It is stated that particles of undissolved test material were visible at the bottom of the test aquaria and that the amount of particles increased with the concentration of the test item. As for the test concentrations 10.0, 14.7, 21.5 and 31.6 mg/L effects were observed, it could not be excluded that these are physical effects due to the undissolved test material. Therefore, these test concentrations could be not evaluated as reliable. The test concentration 6.81 mg/L is the highest test concentration with no effects although undissolved test material was observed.

Based on both available results it can be concluded that N-methyl-N-(C18-(unsaturated)alkanoyl)glycine (EC 701-177-3) will not exhibit short-term effects to fish up to the limit of water solubility.