4-[[4-(aminocarbonyl)phenyl]azo]-N-(2-ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide

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:

From 2001-01-18 to 2001-05-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed according to relevant guideline and compliant to GLP, well documented translation of original report (in Japanese)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Remarks:

This study was conducted in accordance with "Eco-toxicity test Standards" by Japanese Ministry of the Environment.

Test material

Test material information

Constituent 1

Reference substance name:

3-hydroxy-4-[(2-methyl-5-nitrophenyl)azo]-N-phenylnaphthalene-2-carboxamide

EC Number:

229-245-3

EC Name:

3-hydroxy-4-[(2-methyl-5-nitrophenyl)azo]-N-phenylnaphthalene-2-carboxamide

Cas Number:

6448-95-9

Molecular formula:

C24H18N4O4

IUPAC Name:

3-hydroxy-4-[(2-methyl-5-nitrophenyl)diazenyl]-N-phenyl-2-naphthamide

Test material form:

not specified

Details on test material:

Name of test material (as cited in study report): Reproduction Test of ... on Water Fleas (Daphnia magna): C. I. Pigment Red 22 (abbreviated name: PR)
Analytical purity: Over 99%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentration of the test substance in the test solution was measured three times during exposure period (at the preparation of the test solution and before the renewal of the test solution are referred to as one time) for all exposure groups, and analyzed by HPLC.

Test solutions

Vehicle:

yes

Details on test solutions:

After weighing of a necessary amount of test substance using an electronic balance, the same amount of hydrogenated castor oil (HCO-40) was added to the test substance as co-solvent and mixed well. Then, the solution was made up to the volume with dilution water and mixed with a stirrer for 30 minutes to prepare the test solution of the desired nominal concentration.
The prepared test solutions showed strong red color but no visible precipitate was observed. The solution of control and solvent control was observed to be clear and colorless without visible undissolved test substance.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Water fleas
Scientific name: Daphnia magma
Supplier: National Institute for Environmental Studies, Japan
Date of receipt: October 28, 1996
Maintenance after received: Subculture
Acclimation: Acclimation period: February 27, 2001 - March 13, 2001
Mortality of parent daphnids during acclimation period was 0% and ephippia and presence of males were not observed.
Age: Juvenile within 24-hr of age

Condition for acclimation
Water: Dilution water
Loading: 1 daphnids per 50 mL culturing water (100 daphnids / 5 L)
Temperature: 20 +/- 1 deg C
Lighting: Indoor lighting, 16 hr light 18 hr dark
Feeding: Chlorella vulgaris (commercially available condensed chlorella liquid, trade name: raw chlorella-V12, manufacture: Chlorella Industry Co., Ltd.). The amount of organic carbon in 1% suspension was 1192 mg C/L (measured on December, 2000). The amount fed was determined based on this value. The measurement of the amount of organic carbon was contracted out to Koei Techno Company, Limited.
Feed amount: 0.1 - 0.2 mg C (total organic carbon) per daphnid per day.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

No

Test conditions

Hardness:

Total hardness: 60 to 65 mg/L (as CaCO3)

Test temperature:

Water temperature during 21-day exposure: from 20.0 to 20.2 deg C

pH:

7.9 to 8.2. Thus the variation of pH values was below 1.

Dissolved oxygen:

Dissolved oxygen concentrations were between 7.8 to 8.8 mg/L, thus more than 60% of saturation concentration was maintained in all test groups.

Nominal and measured concentrations:

Nominal test item concentration (limit test): 30 mg/L
Measured test item concentrations were beetween 25 and 34 mg/L (for details see tabel under "Any other information on materials and methods incl. tables". The time weighted mean measured concentration during 21 days was determined to be 30 mg/L.
The concentrations of test substance in the control and solvent control were less than quantification limit (0.06 mg/L).

Details on test conditions:

Type of exposure: Semi-Static (water renewal was made after 48 hr exposure)
Duration of exposure: 21-day
Volume of test solution: 80 mL / vessel
Replication: 10 vessel / test group
Number of organisms: 10 daphnids / test group (1 daphnids / vessel)
Temperature: 20 +/- 1 deg C
Lighting: Indoor lightning, 16-hour light / 8-hour dark
Feeding: Chlarella vulgaris
Amount fed: 0.1 - 0.2 mg C (total organic carbon) / daphnid / day

Dilution water
Tap water (Takarazuka city, Hyogo) was dechlorinated with charcoal filter and saturated with oxygen by aeration. Then the water was used as the dilution water after adjusting to the test temperature. The results of water quality measurement are shown in section "Any other information on materials and methods incl. tables". From the results of the water quality measurement and no observed abnormalities in organisms, it was judged that the dilution water can be used as culturing water in the bio-assays.

Test vessels and water bath
Vessel: 100 mL glass beaker
Water bath: Thermostatic water bath for daphnid test

Selection of test concentration
Since the EC50 was more than 100 mg/L in the acute immobilization test, a range-finding study was conducted at 100 mg/L, the highest concentration. The study suggested that reproduction effect observed were caused by the co-solvent contained at the tested concentration. Therefore, the definitive study was conducted as a limit test at 30 mg/L to eliminate the effect caused by co-solvent. As reference, a control group (dilution water only) and a solvent control group, in which co-solvent concentration was 30 mg/L (i.e. the same as for the exposure group) were allocated in the study.

Test operation procedure
The initiation of exposure was defined as the time point at which daphnids were introduced to each vessel using glass pipette after measurement of temperature, dissolved oxygen level (DO) and pH of test solution. The pipetted aliquot of water was less than approximately 1% of the volume of test solution. The daphnids were transferred to new test solution at every water renewal time point and bred for 21 days. The daphnids were fed every day during the exposure period.

Observation of daphnid:
Parent daphnid: Viability, immobilization and abnormal appearance were observed and recorded daily. Dead daphnids were removed from test vessel after observation.
Offspring:
From the appearance of the first offspring, the number of living offspring were counted and removed daily. The presence of dead offspring, aborted egg and ephippia was also observed and recorded daily. The day of first appearance of offspring was recorded as the first brood production day.

Water quality:
The temperature, dissolved oxygen, pH and total hardness of test solution of all test groups (one vessel per test group) were measured three times during exposure period (at the preparation of test solution and before the renewal of test solution are referred to as one time). The measurement at the preparation of test solution was performed using the remaining test solutions after distributing to the test vessels.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

> 30 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

of parental daphnids - no mortality observed

Effect concentrations 2

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 30 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 3

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

30 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

& parental mortality, macroscopic effects

Effect concentrations 4

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 30 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

& parental mortality, macroscopic effects

Details on results:

The percent mortality in the control and solvent control groups were 0% at the termination of exposure (21 days).
The percent mortality at the limit test item concentration of 30 mg/L (treatment group) was also 0% at the termination of exposure (21 d).
The day of first brood production of the parent daphnids in control group was day 7-8 after the initiation of exposure, and it was determined to be within the normal range.
The day of first brood production of the parent daphnids in 30 mg/L exposure group and solvent control group was day 7 after the initiation of exposure, which was similar to the control group.
The mean cumulative number of juveniles produced per adult alive in the control and solvent control group for 21 days were 210.6 and 220.0, respectively. No statistical (t-test) significant difference between the control group and the solvent control group was observed.
The mean cumulative number of juveniles produced per adult alive in 30 mg/L exposure group was 222.7, and no statistical (Aspin-Welch test) significant difference was observed compared with the control group.
There was no difference in size and condition of parent daphnids in 30 mg/L exposure group compared to control group, macroscopically.
The presence of ephippia was not observed in all test groups during the exposure period.
Although aborted eggs were observed occasionally in all groups which produced offspring including the control group, it was considered to be not caused by the test substance since it was also observed in the control group.

Results with reference substance (positive control):

The median effective concentration (EC50) at 48 hr with the reference substance potassium dichromate (reagent grade, Lot No. ACQ2610, Wako Pure Chemical Industries) was 0.64 mg/L, which was comparable to the historical EC50 of 0.38 - 0.94 mg/L, n = 10) obtained at the testing facility since December, 1995.

Reported statistics and error estimates:

No-observed-effect concentration (NOEC) and the lowest-observed-effect concentration (LOEC):
After the calculation of the total numbers of juveniles produced per parent alive in each test group for 21 days, the significance (a = 0.05) between exposure group and control group was analyzed by comparing the mean values of these two groups (t-test).
Based on the results, the highest test concentration with no significant difference (no-observed-effect concentration: NOEC) and the lowest test concentration with significant difference (a = 0.05) (lowest-observed-effect concentration (LOEC) compared to the control group were determined.
Initially, a significance test between control and solvent control group was performed by comparing the mean values of these two groups (t-test). Since no
statistically significant difference was determined between these two groups, the value of control group (dilution water only) was used as reference for statistical tests.

Any other information on results incl. tables

Mean Cumulative Numbers of Juveniles Produced per Adult Alive for 21 Days (Sum F1 per parent):

Nominal						Days
Conc. (mg/L)	0	1	2	3	4	5	6	7	8	9	10
Control	0,0	0.0	0.0	0.0	0.0	0.0	0.0	10.2	16.2	16.2	32.9
Sol. Cont.	0.0	0.0	0.0	0.0	0.0	0.0	0.0	14.6	14.6	14.6	34.8
30	0.0	0.0	0.0	0.0	0.0	0.0	0.0	14.6	14.6	14.6	31.2
Nominal						Days
Conc. (mg/L)	11	12	13	14	15	16	11	18	19	20	21
Control	36.6	40.1	64.8	72.6	97.0	113.4	123.8	151.8	169.2	180.9	210.6
Sol. Cont.	34.8	42.2	73.4	73.4	113.0	118.1	118.1	134.2	165.1	165.1	220.0
30	31.2	63.3	66.7	66.7	118.3	119.0	119.0	165.4	165.4	165.4	222.7

Cumulative Numbers of Juveniles Produced per Adult Alive for 21-Days in Each Test Vessel and Results of Statistical Comparison of the Mean Values (by Aspin-Welch Test)

		Nominal Concentration, mg/L (Measured Concentration*, mg/L)
Vessel No.	Control	Sol. Cont.	30 (30)
1	232	221	213
2	198	231	213
3	227	226	223
4	240	219	246
5	189	218	216
6	191	183	211
7	210	210	227
8	242	240	237
9	190	245	209
10	187	207	232
Mean	210.6	220.0	222.7
S.D.	22.5	17.7	12.5
Inhibition Rate (%)		-4.5	-5.7
Significant Difference**		No	No

* Time-weighted mean measured concentration.

**: Indicates a significant difference by Aspin-Welch Test

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a daphnia magna reproduction test performed according to OECD 211 and compliant to GLP (reliability category 1) with semistatic test design (test medium renewal every 48 hours) no statistically significant effects were observed at the limit test item concentration of 30 mg/L (time weighted mean measured concentration). The following (no)effect concentrations (21 days) were determined:
LC50 (adult mortality) > 30 mg/L (TWA)
LOEC (reproduction, adult mortality, macroscopic effects) > 30 mg/L (TWA)
NOEC (reproduction, adult mortality, macroscopic effects) = 30 mg/L (TWA)
The validity criteria of the guideline were met.

Executive summary:

In a daphnia magna reproduction test performed according to OECD 211 and compliant to GLP (reliability category 1) with semistatic test design (test medium renewal every 48 hours) no statistically significant effects were observed at the limit test item concentration of 30 mg/L (time weighted mean measured concentration).

The high limit test item concentration far above water solubility was achieved with solubilizer (hydrogenated castor oil, HCO-40) applied at equal weight (30 mg/L). A solvent control with the same solubilizer concentration was included in the study.

The percent mortality in the control and solvent control groups were 0% at the termination of exposure (21 days). The percent mortality at the limit test item concentration of 30 mg/L (treatment group) was also 0% at the termination of exposure (21 d).

The day of first brood production of the parent daphnids in control group was day 7-8 after the initiation of exposure, and it was determined to be within the normal range. The day of first brood production of the parent daphnids in 30 mg/L exposure group and solvent control group was day 7 after the initiation of exposure, which was similar to the control group.

The mean cumulative number of juveniles produced per adult alive in the control and solvent control group for 21 days were 210.6 and 220.0, respectively. No statistical (t-test) significant difference between the control group and the solvent control group was observed. The mean cumulative number of juveniles produced per adult alive in 30 mg/L exposure group was 222.7, and no statistical (Aspin-Welch test) significant difference was observed compared with the control group.

There was no difference in size and condition of parental daphnids in 30 mg/L exposure group compared to control group, macroscopically. The presence of ephippia was not observed in all test groups during the exposure period. Although aborted eggs were observed occasionally in all groups which produced offspring including the control group, it was considered to be not caused by the test substance since it was also observed in the control group.

The following (no)effect concentrations (21 days) were determined:

LC50 (adult mortality) > 30 mg/L (geometric mean measured concentration)

LOEC (reproduction, adult mortality, macroscopic effects) > 30 mg/L (geometric mean measured concentration)

NOEC (reproduction, adult mortality, macroscopic effects) = 30 mg/L (geometric mean measured concentration).

The validity criteria of the guideline were met.