ethyl N2-dodecanoyl-.sc.l.sc.-argininate hydrochloride

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 1, 2001 to May 10, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

OECD 203

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Substance: L.A.E.
Purity: 93,2%
Expiry date: March, 2002

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 10, 17.8, 31.6, 56.2 and 100 mg/L
- Sampling method: Samples of 4 mL were taken in the fresh test solutions (including the stock solutions) used for continuous medium renewal at the beginning of the test and each 24 hours thereafter (TO, T24.5, T48.5 and T72.5 hours) as well as in aquarium containing fish just before replacement of these fresh test solutions and at the end of the test (T23.5, T47.5, T71.5 and T96 hours).
- Sample storage conditions before analysis: They were stored at -20°C until analysis.

Vehicle:

no

Details on test solutions:

Test vessels containing fish were filled directly from de test solution containers immediately after preparation. Solutions were continously replace over the 96-hour test period. The pH of the test solutions remained within acceptable limits (between 6 and 9 and in the range ± 0.5 unit of the test water) after preparation and there was no adjustment of pH before addition of the animals.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Source: HB Développement, 69490 Saint Forgeux, France
- Length at study initiation (length definition, mean, range and SD): the animals were an average of 2.91 cm long, ranging from 2.5 to 3.3 cm (based on a measurement of a sample of 31 fish used in the LC50 test).
- Weight at study initiation (mean and range, SD): the mean weight of the fish, based on a sample of ten animals removed at random from the acclimation tank just before the test, was 0.300 g for the preliminary test and 0.291 g for the definitive test, corresponding respectively to a loading of 0.840 g/L (maximum) and 1.16 g/L during the study.
- Method of breeding: During culture, the fish are maintained under flow-through system in water of the same quality and temperature as that to be used in the test. During culture, temperature and water hardness are checked daily while dissolved oxygen concentration and pH are checked at least weekly. During culture, animals were fed TetraMin daily, ceasing 24 hours before the start of the test.
- Feeding during test: Animals were not fed during the test.


ACCLIMATION
- Acclimation period: Yes

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

148-156 mg CaCO3/L

Test temperature:

21.5-22.7 ºC

pH:

7.58-8.3

Dissolved oxygen:

7.4-10.0 mg/L (>60%)

Nominal and measured concentrations:

Nominal (measured) concentrations: 0 ; 10 (3.19); 17.8 (6.60); 31.6 (10.6); 56.2 (56.3) and 100 (91.7) mg/L.

Details on test conditions:

Test solutions were constantly replaced using a replacement rate of 14 times the volume added to each aquarium per day.
TEST SYSTEM
- Test vessel:
- Aeration: was not used during the test. Dilution water was aerated prior to addition of the test item.
- Type of flow-through (e.g. peristaltic or proportional diluter): Test solutions were constantly replaced by a multi-range peristaltic pump.
- Renewal rate of test solution (frequency): 24 hours
- No. of organisms per vessel: 10 fish per vessel
- No. of vessels per concentration (replicates): one replicate
- No. of vessels per control (replicates): one replicate

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Filtered and dechlorinated potable water which id then softened by Permo® water softening system. Half os the matter passes directly into a holding tank attached to the flow-through system while the remaining half passes through a deionizer producing ultra-pure water which is then fed into the holding tank. This result in a 1:1 dilution of potable dechlorinated: deionized water giving a supply of the desirable hardness.
- Alkalinity: pH=6-8.5 and the total hardness is 150±20 mg/L as CaCO3.
- Intervals of water quality measurement: The pH, temperature and dissolved oxygen values of all the groups were measured at the beginning and then every 24 hours thereafter in the limit and definitive tests. Measurements of total hardness in the dilution water were made at the start and the end of the tests.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Light/dark cycle of 16/8 hours is maintained for at least 12 days before the start of the test.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Animals were inspected for mortality and visible abnormalities at each observation time (0, 2, 4, 24, 48, 72 and 96 hours).

TEST CONCENTRATIONS
- Range finding study: Further groups of fish were subjected to dilutions of the limit test solution at 0.01, 0.1, 1 and 10 mg/L for a period of 96 hours.
- Test concentrations: 0, 10, 17.8, 31.6, 56.2 and 100 mg/L.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

8.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

8.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

8.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

8.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

10.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

3.19 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Sub-lethal effects were observed at nominal concentrations of 17.8 and 31.6 mg/L and included animals swimming at the surface of the solution, minor and severe loss of equilibrium, gill bleeding and abnormal respiratory rate.

Reported statistics and error estimates:

Probit method

Sublethal observations / clinical signs:

The concentration of dehydrated N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (L.A.E) measured at the start of the trial could not be maintained in aqueous medium for twenty-four hours where fish were present; the levels measured in samples where fish were not present were adequately maintained. These data suggest that the losses observed were directly attributable to the presence of fish, presumably due to adsorption and/or absorption by the test system.

Table: Cumulative mortality data

Observation time (hours)	Mortality at each nominal concentration (mg/L) n fish out of 10 fish
	Control	10.0	17.8	31.6	56.2	100
0	0	0	0	0	0	0
2	0	1*	0	2	10	10
4	0	1	0	5	10	10
24	0	1	0	10	10	10
48	0	1	0	10	10	10
72	0	1	0	10	10	10
96	0	1	0	10	10	10
* Accidental death (i.e. not due to the test item)

Validity criteria fulfilled:

yes

Remarks:

(No mortality was observed in the control, the conditions were constant during the test, the dissolved oxygen concentration was acceptable, since the substance concentration was not maintained during the test the results are given as measured concent.).

Conclusions:

96-h LC50 of L.A.E in a flow-through test system was 8.36 mg/L for Danio rerio (measured).

Executive summary:

The objective of this study was to assess the acute toxicity of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (L.A.E) dissolved in fresh water to Danio Rerio, in a 96 -hour flow-through test complying with OECD Guideline 203.

Under experimental conditions, the 96 -hour LC50 of L.A.E in a flow-through test system was estimated as 23.7 mg/L based on nominal concentrations and 8.36 mg/L based on measured concentrations for Danio rerio.

Description of key information

Key study: Test method according to OECD 203 and EU method C.1. GLP Study. Under experimental conditions, the 96-hour LC50 of L.A.E in a flow-through test system was estimated as 8.36 mg/L based on measured concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

8.36 mg/L