Butanamide, 2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[3-oxo-, N,N'-bis(o-anisyl and 2,4-xylyl) derivs.

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-06-23 - 1999-07-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study performed according relevant guidelines with plausible and well presented results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OECD Principles on Good Laboratory Practice, (as revised in 1997)

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(2,4-dimethylphenyl)-3-oxobutyramide]

EC Number:

225-822-9

EC Name:

2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(2,4-dimethylphenyl)-3-oxobutyramide]

Cas Number:

5102-83-0

IUPAC Name:

2,2'-[(3,3'-dichlorobiphenyl-4,4'-diyl)didiazene-2,1-diyl]bis[N-(2,4-dimethylphenyl)-3-oxobutanamide]

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

not applicable

Test solutions

Vehicle:

no

Details on test solutions:

A stock dispersion was prepared for each test interval by adding 5 mg of the test substance into a 250 mL beaker. Approximately 150 mL water was added and the mixture was homogenized with an ultra-turrax and by ultrasonification (30 minutes). The mixture was than transferred into a graduated flask, filled up to a volume of 5 L with water for dilution (= 1 mg test substance / L) and stirred for 48 hours with a magnetic stirrer. After a sedimentation phase of 1 hour 100 mL portions of the test dispersion were collected from the middle of the stock dispersion vessel using a glass pipette and subsequently transferred to the test vessels.
The test vessels were preconditioned with test solution to prevent adsorption of the test substance which perhaps would reduce the test concentration below the solubility limit. The test vessels were filled with test solution (nominal concentration 1 mg/L), gently swirled and then the test solution was discarded.
The aqueous phase of the concentration group was present as slight yellowish clear solution during the entire exposure period. There were no visible particulate matters.
Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance is below the analytical detection limit. From the properties of the test substance it could be concluded that the test substance was chemically stable in water over the test intervals of 48 and 72 hours, respectively. The concentration values given in this report are based an the nominal concentration. Due to the very low solubility of the substance it can be estimated, that the actual concentration is smaller or equal to 20 µg/L.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna - Clone 5
Origin: Stock culture of Hoechst Marion Roussel Deutschland GmbH, ProTox
Breeding conditions: 100 mL beakers (height 90 mm, diameter 50 mm), filled with 70 mL test water (according to Elendt, M4) served as culture vessels. One daphnid was used per culture vessel. The animals were used for breeding up to an age of 42 days. The reproduction rate and the state of health of the animals were monitored daily apart from weekends. The animals were fed three times a week with monocellular green algae (Scenedesmus subspicatus).
The supplied diet was based an the amount of organic carbon (C). The ration level is 0.2 mg carbon / day / animal.
During the study the daphnids were fed daily including weekends.
The room was illuminated by fluorescent tubes for 16 hours per day. Daphnids with an age < 24 hours were used for the studies.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

not applicable

Test conditions

Hardness:

Concentration group / Control [mmol Ca(2+) + Mg(2+)]
2.0 - 2.5 / 2.1 - 2.5

Test temperature:

Concentration group / Control [°C]
20.5 to 21.5 / 20.7 to 21.4

pH:

Concentration group / Control
7.8 - 8.3 / 7.8 - 8.1

Dissolved oxygen:

Concentration group / Control [mg/L]
8.6 to 9.4 / 8.6 to 9.3

Salinity:

medium according to Elendt M4

Nominal and measured concentrations:

1 mg/L nominal:
Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance is below the analytical detection limit. From the properties of the test substance it could be concluded that the test substance was chemically stable in water over the test intervals of 48 and 72 hours, respectively. Due to the very low solubility of the substance it can be estimated, that the actual concentration is smaller or equal to 20 µg/L.

Details on test conditions:

The test chambers were 150 mL beakers, each filled with 100 mL test water.

The room temperature was about 21 °C. The room was illuminated by fluorescent tubes in a 16:8 hours day/night cycle.

A measurement of the light intensity at the position of the test vessels in height of the water surface was carried out during the test. The light intensity was 300 lx.

Study groups:
After the test concentrations had been prepared and water parameters were recorded, 1 daphnid was used in each test chamber following randomization plan No. 1999.0207.

The test concentrations were as follows:
Study group 1: Start Jun-23-99, Test chamber No. 1 - 10 Concentration (mg/L): 0 (control)
Study group 2: Start Jun-23-99, Test chamber No. 11 - 20 Concentration (mg/L): 1

Test procedure
The test was conducted in a semistatic system. The daphnids were transferred into fresh prepared test solutions on Monday, Wednesday and Friday. Inspection and feeding of the daphnids took place every 24 hours and involved recording the immobility, the reproduction rate (number of living and dead offspring) and the development of embryos in the brood pouch. Immobile daphnids were removed from the chambers. Those animals not able to swim within 15 seconds after gentle agitation of the test container were considered to be immobile. The pH-value, oxygen content, conductivity and total hardness were measured and recorded once a week at the beginning and at the end of a test interval in one representative test vessel of each concentration group.

Statistics
To find out whether the mean number of living offspring is significantly reduced by substance effects statistical evaluation was carried out. On the premises of a monotonous concentration-effect-relationship the Wilcoxon-Test was applied. The toxic effect is supposed to be a reduction in the mean number of living offspring per parent animal. The concentration group was compared to the control group.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Effect concentrations 2

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

lmmobility:
No immobility occurred in the 1 mg/L (nominal concentration) and in the control group.

Reproduction rate:
No differences in the onset of brood production were observed in the concentration group in comparison to the control. The reproduction rate in the concentration group showed no statistically significant changes in comparison to the control.

Results with reference substance (positive control):

not applicable

Any other information on results incl. tables

Reproduction rate:

	control	1 mg/L
n	10	10
mean of living offspring	126.0	115.4
standard deviation	19.26	17.98
coefficient of variation	15.3	15.6
median	124.0	117.5

n = number of daphnids exposed per concentration

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

- The mortality of the parent animals did not exceed 20 % at the end of the test in the controls. - The mean number of live offspring produced per parent animal surviving at the end of the test was >= 60.

Conclusions:

In this prolonged toxicity study with the test item in Daphnia magna over 21 days the following effect concentrations were obtained:
NOEC (immobility) = 1 mg/L (nominal concentration, solubility limit <= 20 µg/L)
NOEC (reproduction rate) = 1 mg/L (nominal concentration, solubility limit <= 20 µg/L)
No adverse effects of C.I. Pigment Yellow 13 on Daphnia magna were observed at the maximum tested concentration of 1 mg/L (nominal concentration) which was above the solubility limit (<= 20 µg/L) of the test substance in the test water.

Executive summary:

The test item was tested in Daphnia magna (water flea) over 21 days in a semistatic system (GLP, key). Since no effects were expected up to the limit of solubility a limit test with a nominal concentration of 1 mg/L was conducted.

A negative control (0 mg/L) and a nominal concentration of 1 mg/L were tested. The tested nominal concentration is already above the limit of solubility of the test substance in water ( <= 20 µg/L).

The test was conducted under semistatic conditions. The media and the test solutions were renewed every Monday, Wednesday and Friday. For every renewal new test solutions were prepared. This was done by stirring the test substance for 48 hours in the test medium. The aqueous phase of the concentration group was present as a slightly yellowish clear solution during the entire exposure period. There were no visible particulate matters. Water parameters were within the normal range.

Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance is below the analytical detection limit. From the properties of the test substance it could be concluded that the test substance is chemically stable in water over the test intervals of 48 and 72 hours, respectively.

The concentration values given in this report are based on the nominal concentration.

In the control group no mortality occurred and the mean number of living offspring produced per parent animal was 126.0, thus fulfilling the validity criteria for the test.

No immobility occurred in the 1 mg/L (nominal concentration) and in the control group. No difference in the onset of brood production was observed in the concentration group in comparison to the control. The reproduction rate in the concentration group showed no statistically significant changes in comparison to the control.

In this prolonged toxicity study with C.I. Pigment Yellow 13 in Daphnia magna over 21 days the following effect concentrations were obtained:

NOEC (immobility) = 1 mg/L (nominal concentration, solubility limit <= 20 µg/L)

NOEC (reproduction rate) = 1 mg/L (nominal concentration, solubility limit <= 20 pg/L)