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

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 11, 2000 to August 4, 2000

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:

other: Directive 88/302/EC "Biodegradation - Activated Sludge Respiration Inhibition Test"

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The results of preliminary solubility trials showed that suitable suspensions and solutions of L.A.E. could be prepared to allow the addition of aliquots of stock solutions of 0.5 g/l (preliminary test) and 2.5 g/l (definitive test) to be added to test mixtures. Additions of dechlorinated tap water, synthetic sewage and inoculum were made at 15-minute intervals to one-litre beakers (preliminary test) and siliconised (SurfaSilTMP, ierce, UK;2 % v/v solution in acetone), two-litre conical flasks (definitive test).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Method of cultivation: Oakley sewage treatment works, which treats predominantly domestic waste.
- Preparation of inoculum for exposure: Samples were maintained under aerobic conditions until required. Synthetic sewage was added and the mixtures were aereted overnight.
- Pretreatment: On the day of collection, aliquots (25 ml) of the activated sludge were filtered through dried and preweighed Whatman GFC filter papers which were then dried again at approximately 105°C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) was then calculated. Synthetic sewage (50 ml/l) was added and the mixtures were aereted overnight. On the day of the test, the MLSS content of the sludge was determined and adjusted to 4 g/l.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

The water used to prepare the test mixtures was dechlorinated tap water (hardness 200 - 250 mg/l as CaCO3)

Test temperature:

20.2-22.2 ºC

pH:

7.4-7.8

Dissolved oxygen:

Initial measured DO concentration (mg O2/l): 6.5-8.8

Nominal and measured concentrations:

Nominal concentration: 30, 50, 100, 150 and 300 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: Flasks continously agitated on an orbital shaker
- Material, size, headspace, fill volume: 2-litre conical
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water
- Chlorine: Dechlorinated
- Ca/mg ratio: 200-250 mg/l CaCO3

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Rate of oxygen consumption, over a period of approximately 10 minutes or until the dissolved oxygen concentration fell below 2 mgO2/l. Temperature and pH parameters of the samples were measured at the start and end of the test.

TEST CONCENTRATIONS
- Range finding study: 1, 10 and 100 mg/l
- Test concentrations: 30, 50, 100, 150 and 300 mg/l

Reference substance (positive control):

yes

Remarks:

(3,5-dichlorophenol)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

98.5 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL=86.6-112.9

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

< 30 mg/L

Conc. based on:

test mat.

Details on results:

The specific respiration rates of control cultures established at the start and end of the preliminary test were both 30.0 mgO2/g/h. In the definitive test the specific respiration rates of the control at the end of the test series (30.0 mg02/g/h) was 110% of that at the start (27.3 mgO2/g/h).

Results with reference substance (positive control):

Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP. The 3.hour 50% effect concentration (EC50) for 3,5-DCP was calculated by the Moving Average method in the preliminary and definitive tests to be 14.0 mg/l (95% confidence limits 12.2 - 16.3 mg/l) and 6.9 mg/l (95% confidence limits 5.1 - 8.9 mg/l), respectively.

Respiration rate, % inhibition:

Table 1:

LAE (mg/l)	% Inhibition
30	5
50	11
100	51
150	80
300	95
3,5-DCP (mg/1)	% Inhibition
3	33
10	56
12	86

In the definitive test, the final temperature of two mixtures containing 3,5-DCP exceeded the recommended range (18-22°C) for this test type. These deviations were not considered to be significant, nor to have affected the integrity of the test.

Validity criteria fulfilled:

yes

Remarks:

(Variation between the respiration rates of the 2 control samples < 15%. Inhibitory effect of 3,5-dichlorophenol was within the acceptable range).

Conclusions:

The EC50 of the test substance was calculated from the results of the definitive test to be 98.5 mg/L (95% CL 86.6 - 112.9 mg/l).

Executive summary:

According to OECD Guideline 209, Concentration-related inhibition of respiration rates by N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (L.A.E) was seen at test concentrations of 30 mg/l and higher in the definitive test. The highest tested concentration (300 mg/l) caused 95% inhibition.

The EC₅₀ of the test substance was calculated from the results of the definitivetest to be 98.5 mg/l (95% CL 86.6 - 112.9 mg/l). The EC₂₀ and EC₈₀ values were estimated to be 60 and 150 mg/l respectively. The NOEC concentration was estimated to be less than 30 mg/l.

The 3 hour EC₅₀ values for 3,5 -DCP (14.0 mg/l and 6.9 mg/l in the preliminary and definitive test, respectively) fulfilled the validity criterion relating to sensitivity to inhibition (acceptable EC₅₀ range 5 to 30 mg/l), and that relating to the respiration rates in the control (variation not greater than 15%) was also satisfied.

Description of key information

Key study: Test method according to OECD 209. GLP study. The 3h-EC50 of Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (L.A.E) was calculated to be 98.5 mg/l (95% confidence limits 86.6 -112.9 mg/L) and the NOEC concentration was estimated to be less than 30 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

98.5 mg/L

EC10 or NOEC for microorganisms:

30 mg/L