Pentanediamide, N1,N5-dibutyl-2-[(2-ethyl-1-oxohexyl)amino]-, (2S)-

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 October 2005 - 20 October 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentrations of the test item in the test solution were analyzed at commencement and at the end of each 24 hour test period. The four replicates of every concentration plot were pooled.

Vehicle:

no

Details on test solutions:

To obtain a test item concentration as high as possible, the test item solution was prepared by making 1000 mg test material up to 1 L dilution water and stirring vigorously for 21 h at 20 °C under sterile conditions. To avoid possible transfer of particles from the saturated solution into the test vessels, the solution was filtered using a 0.45 µm filter (Sartorius, Minisart®, cellulose acetate). The resulting test item solution was diluted by a factor of 1.5 with dilution water to obtain the other test concentrations. The dilution factor was based on the results of a pre-test.

Dilution water

Reconstituted ISO test water according to the OECD Guideline 202 (annex 3), and ISO 6341 was used:

Calcium chloride solution: 11.76 g CaCI2*2H20 / 1 litre with deionised water
Magnesium sulphate solution: 4.93 g MgSO4*7H2O / 1 litre with deionised water
Sodium bicarbonate solution : 2.59 g NaHCO3 / 1 litre with deionised water
Potassium chloride solution: 0.23 g KCI / 1 litre with deionised water.

All chemicals were of analytical grade.
The conductivity of the distilled or deionised water did not exceed 10 µS x cm^-1.
25 mL each of solutions (a) to (d) was mixed and the total volume was made up to 1 litre with deionised water. The sum of the calcium and magnesium ions in this solution is 2.5 mmol/L. The proportion Ca:Mg ions is 4:1 and Na:K ions 10:1. The acid capacity Ks4.3 of this solution is 0.8 mmol/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna STRAUS, Crustacea, Cladocera.
Age: 4 - 24 hours old.
Origin: Umweltbundesamt, Institut fur Wasser-, Boden- and Lufthygiene, Berlin, Germany. Bred in the laboratory of the Fh-IME.

Breeding and holding conditions:
Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving. Batches of 30 to 50 animals were held at room temperature in approx. 1800 mL purified drinking water. During the week the daphnids were fed daily with an algal suspension (Scenedesmus subspicatus) and Liquizell® (HOBBY) according to the EEC-Guideline. Algae growing in the log-phase were centrifuged and the pellet was resuspended in a few mL of medium. 30 mL of this suspension was given to 1 L Daphnia medium. The water was changed once per week. Newborn daphnids were separated by sieving, the first generation was discarded.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

Not reported

Test temperature:

20.3 to 20.5 °C

pH:

At test start, the pH in the test vessels was between 8.97 and 9.29, after 24 h the pH was between 8.33 and 8.37. At start of the second incubation period the pH in the test vessels was between 8.82 and 9.33, after 24 h the pH was between 8.32 and 8.35.

Dissolved oxygen:

The oxygen saturation in all test concentration plots was between 63 % and 94 % .

Salinity:

Not reported

Nominal and measured concentrations:

The effect (acute immobilization) of the test item on Daphnia magna was tested using five concentrations spaced by a factor of 1.5. The mean measured test item concentrations were: 2.94, 4.44, 6.80, 10.5 and 16.8 mg test item/L.

Details on test conditions:

Dilution water
Reconstituted ISO test water according to the OECD-Guideline was used:

Calcium chloride solution: 11.76 g CaCI2*2H20 / 1 litre with deionised water
Magnesium sulphate solution: 4.93 g MgSO4*7H2O / 1 litre with deionised water
Sodium bicarbonate solution: 2.59 g NaHCO3 / 1 litre with deionised water
Potassium chloride solution: 0.23 g KCI / 1 litre with deionised water.

All chemicals were of analytical grade.

The conductivity of the distilled or deionised water did not exceed 10 µSxcm^-1.
25 mL each of solutions (a) to (d) was mixed and the total volume was made up to 1 litre with deionised water. The sum of the calcium and magnesium ions in this solution was 2.5 mmol/L. The proportion Ca:Mg ions is 4:1 and Na:K ions 10:1. The acid capacity KS4.3 of this solution was 0.8 mmol/L.

Test procedure
The test organisms were exposed to five concentrations of the test item for a period of 48 hours under semi-static conditions. The test concentrations were obtained by dilution of the highest test concentration with dilution water. The concentrations of the test item were chemically analyzed in each concentration plot at the start and end of each 24 hour incubation period.

60 mL glass beakers were used as test vessels. To each beaker 50 mL test solution and 5 daphnids, not older than 24 hours, were added. No feeding and no aeration occurred throughout the test. The controls were held under the same conditions in dilution water. After 24 hours, the daphnids were transferred to freshly prepared test solutions. For every concentration and the control four replicates were prepared.

The temperature during the test ranged from 20.3 to 20.5 °C.
The beakers were covered with glass plates and subjected to a light/dark cycle of 16/8 h with light intensities of not more than 1000 Lux.
At test start before adding the daphnids and at test end, pH-values (WTW Microprocessor pH-Meter pH 196) and oxygen concentrations (WTW Microprocessor Oximeter OXI 196) of pooled samples of the test solutions and control water were measured.
Immobility and abnormal behaviour were recorded after 24 h and 48 h. The animals were considered to be immobile if they are not able to swim within 15 seconds after gentle agitation of the test vessels.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 16.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

10.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

The cumulative immobility of daphnids during the test period of 48 h indicates a concentration-response relationship, ranging from 0 % immobilization at the concentration of 2.94 mg test item/L to 25 % immobilization at the highest concentration of 16.8 mg test item/L. Thus the NOEC was determined to be 10.5 mg/L and the LOEC 16.8 mg test item/L, the highest concentration tested.
After 48 h the EC50 of the test item was determined to be > 16.8 mg test item/L.
The oxygen saturation in all test concentration plots was between 63 % and 94 %. The temperature was between 20.3 and 20.5 °C, the light intensity was between 625 and 663 Lux.
At test start, the pH in the test vessels was between 8.97 and 9.29, after 24 h the pH was between 8.33 and 8.37. At start of the second incubation period the pH in the test vessels was between 8.82 and 9.33, after 24 h the pH was between 8.32 and 8.35.

Results with reference substance (positive control):

The sensitivity of the test clone was checked by using K2Cr2O7 as reference substance. In September 2005 the EC50 was 0.8 mg/L.

Reported statistics and error estimates:

As the immobilization rate was 25 % at the highest test concentration an EC10 and EC50 value could not be calculated. For the assessment of the NOEC the ToxRat programme was used.
Numerical values in the study report were frequently rounded to a smaller degree of precision (number of digits) than were used in the actual calculation. Minor differences in results obtained from calculations with such rounded values in comparison to those obtained with higher precision values are possible. They are however, well within the limits of the experimental accuracy and thus of no practical concern.

Cumulative immobility during the test period of 48 h (5 daphnids per vessel = 20 daphnids per test concentration)

Mean measured concentration Test item mg/L	24 h				48 h				Sum total	Sum (%)
	Beaker 1	Beaker 2	Beaker 3	Beaker 4	Beaker 1	Beaker 2	Beaker 3	Beaker 4
Control	0	0	0	0	0	0	0	1	1	5
2.94	0	0	0	0	0	0	0	0	0	0
4.44	2	0	0	0	2	0	0	0	2	10
6.80	0	0	1	0	0	0	1	1	2	10
10.5	0	0	0	0	0	0	0	0	0	0
16.8	1	2	0	0	1	2	0	2	5	25

 

(No) effect concentrations (mg/L) of the test item (mean measured concentrations)

Test Duration	NOEC	LOEC	EC10	EC50	C.I. of EC50
48 h	10.5	16.8	n.d.	>16.8	n.d.

 

C.I.: 95 % confidence limits

n.d.: not determined due to mathematical reasons

The daphnia acute immobilization test fulfills the validity criteria of the OECD Guideline 202:

- In the control not more than 10 % of the daphnids were immobilized. In the present test 5.0 % of the introduced animals were immobile in the controls.

- The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels. In the present test the oxygen concentration did never fall below 5.4 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The cumulative immobility of daphnids during the test period of 48 h indicates a concentration-response relationship, ranging from 0 % immobilization at the concentration of 2.94 mg test item/L to 25 % immobilization at the highest concentration of 16.8 mg test item/L. Thus the NOEC was determined to be 10.5 mg/L and the LOEC 16.8 mg test item/L, the highest concentration tested.
After 48 h the EC50 of the test item was determined to be > 16.8 mg test item/L.

Executive summary:

A study was performed to evaluate the acute toxicity of the test material to Daphnia magna (AJI-004/4-70) according to OECD Guideline 202.

The test item was tested at mean measured concentrations of 2.94, 4.44, 6.80, 10.5, and 16.8 mg test item/L water under semi static conditions for 48 hours.

The EC50 was determined to be > 16.8 mg test item/L.

The highest concentration without immobilization, also determined as NOEC, was 10.5 mg test item/L.

Description of key information

A study was performed to evaluate the acute toxicity of the test material to Daphnia magna according to OECD Guideline 202.

The cumulative immobility of daphnids during the test period of 48 h indicates a concentration-response relationship, ranging from 0 % immobilization at the concentration of 2.94 mg test item/L to 25 % immobilization at the highest concentration of 16.8 mg test item/L. Thus the NOEC was determined to be 10.5 mg/L and the LOEC 16.8 mg test item/L, the highest concentration tested.

After 48 h the EC50 of the test item was determined to be > 16.8 mg test item/L.

Additional information

A study was performed to evaluate the acute toxicity of the test material to Daphnia magna (AJI-004/4-70) according to OECD Guideline 202.

The test item was tested at mean measured concentrations of 2.94, 4.44, 6.80, 10.5, and 16.8 mg test item/L water under semi static conditions for 48 hours.

The EC50 was determined to be > 16.8 mg test item/L.

The highest concentration without immobilization, also determined as NOEC, was 10.5 mg test item/L.