4-[(2,5-dichlorophenyl)azo]-3-hydroxy-N-(2-methoxyphenyl)naphthalene-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:

2010-01-13 to 2010-02-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study meets requirements of OECD Guideline 211 and GLP requirements with no deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-hydroxy-N-(o-tolyl)-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

EC Number:

229-440-3

EC Name:

3-hydroxy-N-(o-tolyl)-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

Cas Number:

6535-46-2

Molecular formula:

C24H16Cl3N3O2

IUPAC Name:

3-hydroxy-N-(2-methylphenyl)-4-[(2,4,5-trichlorophenyl)diazenyl]-2-naphthamide

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Name of test material (as cited in study report): (no report title): Pigment Red 112
Analytical purity: 99.8 %

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

No sampling for determination of the test item

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution (1 mg/l test item was weighed out) was prepared with dilution water two days prior to application.
The stock solution was shaken with 20 rpm for 48 h (rotating shaker 3040, GFL). Undissolved particles were removed by membrane filtration (0.45 µm, RC, MACHEREY-NAGEL).
- Eluate: Dilution water
- Differential loading: Saturated solution as limit concentration
- Controls: 10 replicates of dilution water without test item

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Frequency: daily


ACCLIMATION
- Acclimation period: 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: During acclimation the daphnids were not fed
- Feeding frequency: None
- Health during acclimation (any mortality observed): Healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The parent animals were removed from the culture medium and the juveniles collected over a sieve and flushed into fresh medium

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Test conditions

Hardness:

Total Hardness as CaCO3 [mg/L]
I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Jan. 13th Jan. 15th Jan. 20th Jan. 22nd Jan. 27th Jan. 29th
Saturated Solution 167 167 182 185 176 173
Control 180 170 164 189 180 175

Test temperature:

Temperature [°C]
I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Jan. 13th Jan. 15th Jan. 20th Jan. 22nd Jan. 27th Jan. 29th
Saturated Solution 20.3 21.9 19.9 20.4 20.0 20.5
Control 20.0 21.5 19.1 20.6 19.3 21.0

pH:

pH-Values
I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Jan. 13th Jan. 15th Jan. 20th Jan. 22nd Jan. 27th Jan. 29th
Saturated Solution 7.51 7.42 7.71 7.46 7.43 7.37
Control 7.92 7.47 7.93 7.14 7.87 7.18

Dissolved oxygen:

Dissolved Oxygen Concentration [mg O2/L]
I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Jan. 13th Jan. 15th Jan. 20th Jan. 22nd Jan. 27th Jan. 29th
Saturated Solution 8.14 6.87 7.79 6.78 8.17 8.74
Control 8.36 6.57 8.49 7.29 9.17 6.07

Salinity:

Not measured, freshwater

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency): 3 x per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark per day
- Light intensity: Max. 20 µE*m-2*s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Adult mortality: daily
- Number of juveniles: daily
- Stillborn juveniles and aborted eggs: daily
- Appearance of first brood
- Intrinsic rate of natural increase: test end
- Growth (total length and dry weight): test end


RANGE-FINDING STUDY
- Test concentrations: 1:1 (Saturated solution with a nominal loading 5 mg/L), 1:10, 1:100

Immobilization Rates [%] of the Preliminary Acute Immobilization Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Dilution level
of the
saturated solution IMMOBILIZATION [%]
24 h 48 h
Replicate Replicate
1 2 MV 1 2 MV
1:1# 0 0 0 0 0 0
1:10 0 0 0 0 0 0
1:100 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

1 other: saturated solution (with the nominal loading of 1.00 mg/L)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Details on results:

- Mortality of parent animals: No adult mortality or immobilization of parental daphnids was observed at the control group and at the saturated solution
- No. of offspring produced per day per female: At the control group and the saturated solution five broods were released during the test period by all of the daphnids. The mean number of offspring alive produced per parent animal surviving at the end of the test was 116 juveniles at the control group and 112 juveniles at the saturated solution after 21 days. The reproductive output at the limit concentration is comparable to the control group (t-test, p = 0.05).
- Type and number of morphological abnormalities: Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was < 1 %, which is seen to be not biologically significant. At the control no dead juveniles or aborted eggs were observed.
- Number of males and females (parental): No males were observed in either the control or the test groups during the test.
- Time to first brood release or time to hatch: The first day of appearance of juveniles at the test and control group was day 8

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.20 (CI 1.08 - 1.34)

Reported statistics and error estimates:

The NOEL and LOEL for reproduction as measured by the intrinsic rate of natural increase and total living neonates per surviving female as well as adult survival were determined directly from the results. Significant deviations were determined in comparison to control using statistical standard procedures as normality test, equal variance test and t-test.
Prior to running a t-test a normality test and an equal variance test were performed. P-values for both normality and equal variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a=0.05.
For the determination of significant deviations for the reproduction rates and the intrinsic rates of natural increase t-test (p = 0.05)
The coefficients of variation around the mean number of living offspring produced per parent at the control group and the saturated solution were evaluated.

EC-values and statistical EC50-value was only calculated for the reference item by sigmoidal dose-response regression. The confidence interval for the EC50-value was calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism.

Any other information on results incl. tables

Number of Juveniles in the Control and Test Group after 21 d

Nominal Loading of the Saturated Solution [mg/L]												No.	Mean No. of Juveniles			Compared
	Number of Juveniles in Replicate No.										Total	of			CV	versus
											No.	Parents	per Parent			Control
												prod. Juv.	producing Juveniles			Red.
											å	N	MV ± SD		[%]	[%]
1.00	105	110	127	117	104	119	115	95	110	121	1123	10	112 ±	9	8	3
Control	153	95	97	123	140	124	124	103	97	104	1160	10	116 ±	20	17	—

Mortality [%] of the Adult Daphnids after 7, 14 and 21 d of Exposure

                        (n = 10)

Nominal Loading of the Saturated Solution [mg/L]	Mortality [%]
	7 days	14 days	21 days
1.00	0	0	0
Control	0	0	0

Stillborn Juveniles and Aborted Eggs after 21 d

Nominal Loading of the Saturated Solution [mg/L]	Number of		Total No.		Mean Stillborn Juveniles and Aborted Eggs per Parent alive
	Stillborn	Aborted
	Juveniles	Eggs	å	N	MV ± SD
1.00	0	5	5	10	0.50 ± 0.53
Control	0	0	0	10	0 ± 0

First Appearance of Living Juveniles in the Individual Groups

Nominal Loading of the Saturated Solution [mg/L]	Day of First Appearance of Living Juveniles										First
	in Replicate No.										Appearance
	1	2	3	4	5	6	7	8	9	10	Mean Day
1.00	8	8	8	8	8	8	8	8	8	8	8.0
Control	8	8	8	8	8	8	8	8	8	8	8.0

Total Length and Dry Weight of the Parental Daphnids

Nominal Loading of the Saturated Solution [mg/L]	Total Length of the Parental Daphnids [mm]										MV	N	Dry Weight [mg]
	Replicate No.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
1.00	4.75	5.25	5.25	5.25	5.00	4.75	5.00	4.50	4.75	5.00	4.95	10	6.6	0.66
Control	5.50	5.50	5.00	5.50	5.50	5.00	5.25	5.00	4.75	5.25	5.23	10	7.7	0.77

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

At the saturated solution of the test item with a nominal loading of 1.00 mg/L no biologically or statistically significant effects were determined. The No Observed Effect Loading (NOEL) and the Lowest Observed Effect Loading (LOEL) were assessed at:
NOEL: Saturated Solution (with the nominal loading of 1.00 mg/L)
LOEL: > Saturated Solution (with the nominal loading of 1.00 mg/L)
All effect values given are based on the saturated solution of the test item with the nominal loading of 1.00 mg/L.

Executive summary:

The Daphnia magna Reproduction Test (Semi-Static, 21 d) of the test item was conducted as a limit test with the saturated solution* according to OECD 211 (2008) from 2010-01-13 to 2010-02-04 with the definitive exposure phase between 2010-01-13 to 2010-02-03 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1, D-31157 Sarstedt, Germany.
Test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held were used for the limit concentration and control. At test start they were 2 to 24 h old. The test method was semi-static. The test solutions were renewed 3 times per week. Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects on parameters such as occurrence of aborted eggs and stillborn juveniles, time of production of first brood, adult mortality, intrinsic rate of natural increase, dry body weight and length of the parent animals.
A specific analytic of the test item was not carried out, due to the low solubility of the test item in water. All results given are based on the saturated solution* with the nominal loading of 1mg/L.

·   The average number of juveniles per parental daphnid in the control group was 116 and 112 at the saturated solution* after 21 days. The reproductive output at the limit concentration is comparable to the control group (t-test, p = 0.05).

·   The coefficient of variation of the number of living offspring produced per parent was 17 % at the control group and 8 % at the saturated solution*.

·   The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The IR of the saturated solution* was comparable to the control group (t-test, p = 0.05).

·   No adult mortality or immobilization of parental daphnids was observed at the control group and at the saturated solution*.

·   Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs was lower than 1 % at the saturated solution, which is seen to be not biologically significant. At the control no dead juveniles or aborted eggs were observed.

Five broods were observed during the test period at all surviving parental daphnids. The first brood was released till day 8 by all parent animals of the control and the saturated solution.

·   The mean dry body weight and total body lengths of all parental daphnids of the test and control group determined at the end of the study were comparable.
The mean dry body weight of the parental daphnids was 0.77 mg per daphnid at the control group and 0.66 mg per daphnid at the saturated solution. The mean body length of the parental daphnids at the saturated solution was 4.95 mm per daphnid and 5.23 mm per daphnid at the control group.

·   No malesand ephippia (winter eggs) were observed at control or test groups.

A summary of all test item related effects is given in Table 1. There was no biologically or statistically significant effect determined at the saturated solution of the test item.

Table 1:    Summary of all Test Item Related Effects

                   (Based on the saturated solution of Pigment Red 112with the nominal loading of 1 mg/L)

Effects		Test item	Significance
	Control	Saturated Solution (Nominal Loading 1 mg/L)
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	116 ± 20	112 ± 9	no
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent	17	8	no
Intrinsic Rate of Natural Increase	0.48	0.48	no
Appearance of First Brood [Mean Day]	8.0	8.0	no
Number of Broods	5	5	no
Stillborn Juveniles and Aborted Eggs	0	5	no
Adult Mortality after 21 days [%]	0	0	no
Parent Animals: Mean Dry Weight [mg]	0.77	0.66	no
Parent Animals: Mean Body Length [mm]	5.23	4.95	no
NOELReproduction	Saturated Solution* (Nominal Loading 1 mg/L)
LOELReproduction	> Saturated Solution* (Nominal Loading 1 mg/L)

The water quality parameters (pH-value, dissolved oxygen, water hardness and temperature) were determined to be within the acceptable limits.

·          In order to prove the validity of the test system and test conditions at the test facility, an acute immobilization test according to DIN 38412 L 11 was carried out with potassium dichromateas reference item once per month.
The EC₅₀ of the reference item at 1.20 mg/L after 24 h was within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) for daphnids clone 5 cultured in Elendt M4 medium. The EC₅₀-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.