N-(4-chloro-2,5-dimethoxyphenyl)-3-hydroxy-4-[(E)-2-[2-methoxy-5-(phenylcarbamoyl)phenyl]diazen-1-yl]naphthalene-2-carboxamide; N-(5-chloro-2-methylphenyl)-3-hydroxy-4-[(E)-2-[2-methoxy-5-(phenylcarbamoyl)phenyl]diazen-1-yl]naphthalene-2-carboxamide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

No sampling for determination of the test item

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 (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

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

Not observed

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

Duration:

21 d

Dose descriptor:

NOELR

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Remarks on result:

not determinable

Remarks:

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

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.

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

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.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Remarks:

Pigment Red 022

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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)

Justification for type of information:

See Rationale and Justification for the Analogue Read-Across Approach for the registration of the Nanoform of Pigment Red 184 (Chapter 13)

Reason / purpose for cross-reference:

read-across source

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.

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.

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 (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.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

No

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

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

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 30 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

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

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.

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

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.

Reference 3

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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.

Justification for type of information:

See Rationale and Justification for the Analogue Read-Across Approach for the registration of the Nanoform of Pigment Red 184 (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

No sampling for determination of the test item

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 (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

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

Not observed

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

Duration:

21 d

Dose descriptor:

NOELR

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and reproduction

Remarks on result:

not determinable

Remarks:

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

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.

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

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.

Reference 4

Endpoint:

long-term toxicity to aquatic invertebrates

Remarks:

Pigment Red 022

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)

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.

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.

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 (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.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

No

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

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

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 30 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

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

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.

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

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.

Description of key information

PR022

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.

 

PR112

The test substance was tested in a daphnia magna reproduction test performed according to OECD 211 and compliant to GLP (reliability category 1). 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.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

> 1 mg/L