2-[(4-methoxy-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

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:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2011-11-14 to 2011-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed according to relevant guidelines and compliant to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetzt and directive 88/320/EEC

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Saturated solution as limit test concentration
- Sampling method:
Analytical evaluation of the saturated solution of test item and the control was carried out by HPLC-DAD in the fresh media (0 h) and old media (24 h) every 7 days. For the old media, separate replicates without test organisms were prepared and incubated under test conditions.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 C° until sample preparation, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solutions (1 mg/L test item was weighed out) were prepared with dilution water two days prior to application / water
renewal. The stock solution was stirred for 48 hours at room temperature with a magnetic stirrer (approximately 1100 rpm). Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). Due to the test item properties centrifugation was not sufficient. This was checked in a preliminary test.
- Eluate: Dilution water
- Differential loading: Saturated solution (1mg/L) as limit test 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]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
2011-11-16 2011-11-17 2011-11-23 2011-11-24 2011-11-30 2011-12-01
1.00
(Saturated solution) 164 164 166 164 176 169
Control 176 162 176 169 176 164

Test temperature:

Temperature [°C]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
2011-11-16 2011-11-17 2011-11-23 2011-11-24 2011-11-30 2011-12-01
1.00
(Saturated solution) 21.5 20.8 21.2 21.6 20.4 21.2
Control 20.1 20.6 20.3 21.5 20.8 21.1

pH:

pH-Values
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
2011-11-16 2011-11-17 2011-11-23 2011-11-24 2011-11-30 2011-12-01
1.00
(Saturated solution) 8.14 8.02 7.68 7.56 7.81 7.38
Control 7.92 7.80 7.79 7.49 7.95 7.46

Dissolved oxygen:

Dissolved Oxygen Concentration [mg O2/L]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
2011-11-16 2011-11-17 2011-11-23 2011-11-24 2011-11-30 2011-12-01
1.00
(Saturated solution) 5.71 9.04 6.28 8.50 5.91 7.69
Control 8.84 8.53 8.76 7.49 8.85 7.34

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to Any other information on materials and methods incl. tables

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): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control and pH-control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to ELENDT (1990), modified to a total hardness of > 140 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
Immobilization Rates in the Preliminary Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item Concentration

[mg/L] IMMOBILIZATION [%]
24 hours 48 hours
Replicates Replicates
1 2 MV 1 2 MV
1.00
(Saturated solution) 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:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and mortality

Remarks on result:

other: Due to the very poor solubility of the test item, quantitative analytical determination by HPLC-DAD was precluded (below LOQ).

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and mortality

Remarks on result:

other: Due to the very poor solubility of the test item, quantitative analytical determination by HPLC-DAD was precluded (below LOQ).

Details on results:

- Mortality of parent animals:
No adult mortality or immobilization of parental daphnids was observed in control and the saturated solution with 1.00 mg test item/L
- No. of offspring produced per day per female:
In the control and the saturated test item solution (limit concentration), five broods were released during the test period by all daphnids, respectively. The first brood was released on days 8 and 9 by all parent daphnids in the control and test group.
The mean number of alive offspring produced per parent daphnid surviving the test was 126 in the control and also in the saturated test item solution with 1.00 mg test item/L after 21 days. The reproductive output of daphnids in the saturated test item solution (limit concentration) was comparable to the output of daphnids in the control (MANN-WHITNEY Rank Sum Test, p < 0.05). The coefficient of variation of the number of living offspring per parent was 9 % in the control and 4 % in the saturated solution with 1.00 mg test item/L, respectively
- Body length and weight of parent animals:
At the end of the test, the total body lengths and dry weight of all surviving parental daphnids of the test and the control group were determined as described in the table under "Remarks on results".
- Type and number of morphological / behavioural abnormalities:
Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was < 1 % in the saturated solution, which is seen to be not biologically significant. In the control no dead juveniles or aborted eggs were observed.
- Time to first brood release or time to hatch:
The first brood was released from all parent daphnids in all treatments by day 9

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.60 (CI 1.41 - 1.82) mg/L

Reported statistics and error estimates:

The NOEC and LOEC for reproduction as measured by the intrinsic rate of natural increase and total living neonates per surviving female as well as adult survival was derived directly from the test data.
Significant deviations were determined in comparison to the control using statistical standard procedures as Normality Test, Equal Variance Test and t-test.
Prior to running an analysis of variance test, a normality test and an equal variance test were performed. P-values of the normality and equal variance tests 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 of the reproduction rates MANN-WHITNEY Rank Sum Test (p < 0.05) was used. For the determination of significant deviations of the intrinsic rates of natural increase and the body lengths t-test (p = 0.05) was used.
The coefficients of variation of alive offspring produced per parent were calculated for the control and the saturated test item solution.

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

Nominal Test Item Concentration [mg/L]												No. of Parents producing Juveniles	Mean No. of Juveniles per Parent producing Juveniles			Compared versus Control
	Number of Juveniles in Replicate No.										Total
											No.
														CV		Red.		Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD	[%]		[%]
1.00 (Saturated solution)	133	125	124	128	129	129	118	125	126	120	1257	10	126 ± 4	4		0		no
Control	125	125	122	123	119	126	114	117	153	136	1260	10	126 ± 11		9		—

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

                 (n = 10)

Nominal Test Item Concentration [mg/L]	Mortality [%]
	7 days	14 days	21 days
1.00 (Saturated solution)	0	0	0
Control	0	0	0

Relative Number of Dead (Stillborn + Aborted Eggs) to Total Number of Juveniles

Nominal Test Item Concentration [mg/L]	Number of Juveniles			Percentage of Dead Juveniles* [%]
	Dead	Alive	Total
1.00 (Saturated solution	3	1257	1260	<1
Control	0	1260	1260	0

Dead = Aborted eggs + stillborn juveniles

Total = Deasd + alive juveniles

* = Related to the total number of juveniles

First Appearance of Living Juveniles in the Individual Groups

Nominal Test Item Concentration [mg/L]	Day of First Appearance of Living Juveniles										First Appearance Mean Day
	in Replicate No.
	1	2	3	4	5	6	7	8	9	10
1.00 (Saturated solution)	8	8	9	9	9	9	9	9	9	8	8.7
Control	8	9	8	8	8	8	8	9	9	9	8.4

Total Length and Dry Weight of the Parental Daphnids

Nominal Test Item Concentration [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 (Saturated solution)	5.00	5.25	5.75	5.75	5.25	5.25	5.25	5.50	5.50	5.50	5.40	10	11.0	1.10
Control	5.25	5.25	5.50	5.50	5.50	5.50	5.50	5.50	5.75	5.75	5.50	10	9.3	0.93

N= number of parental daphnids alive

Validity criteria fulfilled:

yes

Conclusions:

A semi-static (daily renewal) 21-day Daphnia magna reproduction test was performed with the test item as a limit test with the saturated solution. Due to the very poor solubility of the test item, qantitative analytical determination by HPLC-DAD was precluded (below LOQ). At the saturated test item solution with 1.00 mg test item/L no biologically or statistically significant effects were determined. The No Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration (LOEC) were as follows:
NOEC : 1.00 mg/L
LOEC : > 1.00 mg/L
All provided effect values are based on the nominal concentration of the test item.

Executive summary:

The Daphnia magna Reproduction Test (Semi-Static with daily renewal, 21 d) ot the test item was conducted as a limit test with the saturated solution with 1.00 mg/L test item according to OECD 211 (2008).
The selected test organism was Daphnia magna STRAUS (Clone 5). Ten daphnids individually held were used for the saturated test item solution and the control, respectively. At test start the daphnids were 2 to 24 h old. The test method was semi-static with a daily renewal of the test solutions. The aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects, including 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 daphnids.
The tested saturated solution of the test item and the control were analytically verified by HPLC-DAD of samples on days 0, 7, 14 (fresh media, 0 hours) and on days 1, 8, 15 (old media, 24 hours).

Due to the very low solubility of the test item it was not possible to measure the test item concentrations in samples of the fresh (0 hours) and the old media (24 hours) of the saturated solution and the control. The results given are based on the nominal concentration of 1.00 mg/L of the test item.

·   The average values of juveniles per parental daphnid were 126 for the control and also for the saturated test item solution with 1.00 mg test item/L after 21 days. The reproductive output of daphnids in the saturated test item solution* (limit concentration) was comparable to the output of daphnids in the control (Mann-Whitney Rank Sum Test, p  0.05).

·   The coefficient of variation of the number of living offspring produced per parent was 9 % for the control and 4 % for the saturated test item solution, respectively.

·   The intrinsic rates of natural increase (IR) of the surviving parent daphnids accounting for generation time and number of offspring were calculated as a measure for 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 in the control and 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 % in the saturated solution, which is seen to be not biologically significant. In the control no dead juveniles or aborted eggs were observed.

Five broods, respectively, were observed during the test period for all parental daphnids. The first brood was released from all parent daphnids in all treatments by day 9.

·   The mean dry body weight and total body lengths of all parental daphnids of the test and the control group were determined at the end of the study.
The mean dry body weight of the parental daphnids was 0.93 mg per daphnid for the control and 1.10 mg per daphnid for the saturated test item solution.
The mean body length of the parental daphnids was 5.50 mm per daphnid for the control and 5.40 mm per daphnid for the saturated test item solution.

·   No males and ephippia (winter eggs) were observed in the respective control or test groups.

A summary of all test item related effects is given in Table 1. No biologically or statistically significant effects could be determined in the saturated test item solution with 1.00 mg/L test item when compared to the control.

Table 1:    Summary of all Test Item Related Effects

                   (Based on the nominal concentration of the test item of 1.00 mg/L)

		Test Item
Effects	Control	Nominal Concentration 1.00 mg/L	Significance
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	126 ± 11	126 ± 4	no
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent	9	4	no
Intrinsic Rate of Natural Increase (Mean ± SD)	0.47 ± 0.02	0.47 ± 0.02	no
Appearance of First Brood (Mean Day ± SD)	8.4 ± 0.5	8.7 ± 0.5	—
Number of Broods	5	5	—
Stillborn Juveniles and Aborted Eggs	0	3	—
Adult Mortality after 21 Days [%]	0	0	no
Parent Daphnids: Mean Dry Weight [mg]	0.93	1.10	—
Parent Daphnids: Mean Body Length [mm] ± SD	5.50 ± 0.17	5.40 ± 0.24	no
NOEC	1.00 mg/L

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

- To assure the validity of the test system and the test conditions at the test facility, an acute immobilization test according to DIN 38412 L 11 is being carried out with potassium dichromate as reference item once per month. This reference test is required by the test guideline OECD 211 (2008).
The EC₅₀ of the reference item of 1.60 mg/L (95 % confidence limits: 1.41 - 1.82 mg/L) after 24 h is 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

Data waiving:

other justification

Justification for data waiving:

other:

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:

2011-11-14 to 2011-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed according to relevant guidelines and compliant to GLP

Justification for type of information:

See chapter 13: Rationale and Justification for the Analogue Read-Across Approach used for the Registration of C.I. Pigment Yellow 65

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 (incl. QA statement)

Remarks:

according to German Chemikaliengesetzt and directive 88/320/EEC

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Saturated solution as limit test concentration
- Sampling method:
Analytical evaluation of the saturated solution of test item and the control was carried out by HPLC-DAD in the fresh media (0 h) and old media (24 h) every 7 days. For the old media, separate replicates without test organisms were prepared and incubated under test conditions.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 C° until sample preparation, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solutions (1 mg/L test item was weighed out) were prepared with dilution water two days prior to application / water
renewal. The stock solution was stirred for 48 hours at room temperature with a magnetic stirrer (approximately 1100 rpm). Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). Due to the test item properties centrifugation was not sufficient. This was checked in a preliminary test.
- Eluate: Dilution water
- Differential loading: Saturated solution (1mg/L) as limit test 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]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
2011-11-16 2011-11-17 2011-11-23 2011-11-24 2011-11-30 2011-12-01
1.00
(Saturated solution) 164 164 166 164 176 169
Control 176 162 176 169 176 164

Test temperature:

Temperature [°C]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
2011-11-16 2011-11-17 2011-11-23 2011-11-24 2011-11-30 2011-12-01
1.00
(Saturated solution) 21.5 20.8 21.2 21.6 20.4 21.2
Control 20.1 20.6 20.3 21.5 20.8 21.1

pH:

pH-Values
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
2011-11-16 2011-11-17 2011-11-23 2011-11-24 2011-11-30 2011-12-01
1.00
(Saturated solution) 8.14 8.02 7.68 7.56 7.81 7.38
Control 7.92 7.80 7.79 7.49 7.95 7.46

Dissolved oxygen:

Dissolved Oxygen Concentration [mg O2/L]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
2011-11-16 2011-11-17 2011-11-23 2011-11-24 2011-11-30 2011-12-01
1.00
(Saturated solution) 5.71 9.04 6.28 8.50 5.91 7.69
Control 8.84 8.53 8.76 7.49 8.85 7.34

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to Any other information on materials and methods incl. tables

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): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control and pH-control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to ELENDT (1990), modified to a total hardness of > 140 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
Immobilization Rates in the Preliminary Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item Concentration

[mg/L] IMMOBILIZATION [%]
24 hours 48 hours
Replicates Replicates
1 2 MV 1 2 MV
1.00
(Saturated solution) 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:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and mortality

Remarks on result:

other: Due to the very poor solubility of the test item, quantitative analytical determination by HPLC-DAD was precluded (below LOQ).

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and mortality

Remarks on result:

other: Due to the very poor solubility of the test item, quantitative analytical determination by HPLC-DAD was precluded (below LOQ).

Details on results:

- Mortality of parent animals:
No adult mortality or immobilization of parental daphnids was observed in control and the saturated solution with 1.00 mg test item/L
- No. of offspring produced per day per female:
In the control and the saturated test item solution (limit concentration), five broods were released during the test period by all daphnids, respectively. The first brood was released on days 8 and 9 by all parent daphnids in the control and test group.
The mean number of alive offspring produced per parent daphnid surviving the test was 126 in the control and also in the saturated test item solution with 1.00 mg test item/L after 21 days. The reproductive output of daphnids in the saturated test item solution (limit concentration) was comparable to the output of daphnids in the control (MANN-WHITNEY Rank Sum Test, p < 0.05). The coefficient of variation of the number of living offspring per parent was 9 % in the control and 4 % in the saturated solution with 1.00 mg test item/L, respectively
- Body length and weight of parent animals:
At the end of the test, the total body lengths and dry weight of all surviving parental daphnids of the test and the control group were determined as described in the table under "Remarks on results".
- Type and number of morphological / behavioural abnormalities:
Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was < 1 % in the saturated solution, which is seen to be not biologically significant. In the control no dead juveniles or aborted eggs were observed.
- Time to first brood release or time to hatch:
The first brood was released from all parent daphnids in all treatments by day 9

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.60 (CI 1.41 - 1.82) mg/L

Reported statistics and error estimates:

The NOEC and LOEC for reproduction as measured by the intrinsic rate of natural increase and total living neonates per surviving female as well as adult survival was derived directly from the test data.
Significant deviations were determined in comparison to the control using statistical standard procedures as Normality Test, Equal Variance Test and t-test.
Prior to running an analysis of variance test, a normality test and an equal variance test were performed. P-values of the normality and equal variance tests 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 of the reproduction rates MANN-WHITNEY Rank Sum Test (p < 0.05) was used. For the determination of significant deviations of the intrinsic rates of natural increase and the body lengths t-test (p = 0.05) was used.
The coefficients of variation of alive offspring produced per parent were calculated for the control and the saturated test item solution.

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

Nominal Test Item Concentration [mg/L]												No. of Parents producing Juveniles	Mean No. of Juveniles per Parent producing Juveniles			Compared versus Control
	Number of Juveniles in Replicate No.										Total
											No.
														CV		Red.		Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD	[%]		[%]
1.00 (Saturated solution)	133	125	124	128	129	129	118	125	126	120	1257	10	126 ± 4	4		0		no
Control	125	125	122	123	119	126	114	117	153	136	1260	10	126 ± 11		9		—

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

                 (n = 10)

Nominal Test Item Concentration [mg/L]	Mortality [%]
	7 days	14 days	21 days
1.00 (Saturated solution)	0	0	0
Control	0	0	0

Relative Number of Dead (Stillborn + Aborted Eggs) to Total Number of Juveniles

Nominal Test Item Concentration [mg/L]	Number of Juveniles			Percentage of Dead Juveniles* [%]
	Dead	Alive	Total
1.00 (Saturated solution	3	1257	1260	<1
Control	0	1260	1260	0

Dead = Aborted eggs + stillborn juveniles

Total = Deasd + alive juveniles

* = Related to the total number of juveniles

First Appearance of Living Juveniles in the Individual Groups

Nominal Test Item Concentration [mg/L]	Day of First Appearance of Living Juveniles										First Appearance Mean Day
	in Replicate No.
	1	2	3	4	5	6	7	8	9	10
1.00 (Saturated solution)	8	8	9	9	9	9	9	9	9	8	8.7
Control	8	9	8	8	8	8	8	9	9	9	8.4

Total Length and Dry Weight of the Parental Daphnids

Nominal Test Item Concentration [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 (Saturated solution)	5.00	5.25	5.75	5.75	5.25	5.25	5.25	5.50	5.50	5.50	5.40	10	11.0	1.10
Control	5.25	5.25	5.50	5.50	5.50	5.50	5.50	5.50	5.75	5.75	5.50	10	9.3	0.93

N= number of parental daphnids alive

Validity criteria fulfilled:

yes

Conclusions:

A semi-static (daily renewal) 21-day Daphnia magna reproduction test was performed with the test item as a limit test with the saturated solution. Due to the very poor solubility of the test item, quantitative analytical determination by HPLC-DAD was precluded (below LOQ). At the saturated test item solution with 1.00 mg test item/L no biologically or statistically significant effects were determined. The No Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration (LOEC) were as follows:
NOEC : 1.00 mg/L
LOEC : > 1.00 mg/L
All provided effect values are based on the nominal concentration of the test item.

Executive summary:

The Daphnia magna Reproduction Test (Semi-Static with daily renewal, 21 d) ot the test item was conducted as a limit test with the saturated solution with 1.00 mg/L test item according to OECD 211 (2008).
The selected test organism was Daphnia magna STRAUS (Clone 5). Ten daphnids individually held were used for the saturated test item solution and the control, respectively. At test start the daphnids were 2 to 24 h old. The test method was semi-static with a daily renewal of the test solutions. The aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects, including 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 daphnids.
The tested saturated solution of the test item and the control were analytically verified by HPLC-DAD of samples on days 0, 7, 14 (fresh media, 0 hours) and on days 1, 8, 15 (old media, 24 hours).

Due to the very low solubility of the test item it was not possible to measure the test item concentrations in samples of the fresh (0 hours) and the old media (24 hours) of the saturated solution and the control. The results given are based on the nominal concentration of 1.00 mg/L of the test item.

·   The average values of juveniles per parental daphnid were 126 for the control and also for the saturated test item solution with 1.00 mg test item/L after 21 days. The reproductive output of daphnids in the saturated test item solution* (limit concentration) was comparable to the output of daphnids in the control (Mann-Whitney Rank Sum Test, p  0.05).

·   The coefficient of variation of the number of living offspring produced per parent was 9 % for the control and 4 % for the saturated test item solution, respectively.

·   The intrinsic rates of natural increase (IR) of the surviving parent daphnids accounting for generation time and number of offspring were calculated as a measure for 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 in the control and 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 % in the saturated solution, which is seen to be not biologically significant. In the control no dead juveniles or aborted eggs were observed.

Five broods, respectively, were observed during the test period for all parental daphnids. The first brood was released from all parent daphnids in all treatments by day 9.

·   The mean dry body weight and total body lengths of all parental daphnids of the test and the control group were determined at the end of the study.
The mean dry body weight of the parental daphnids was 0.93 mg per daphnid for the control and 1.10 mg per daphnid for the saturated test item solution.
The mean body length of the parental daphnids was 5.50 mm per daphnid for the control and 5.40 mm per daphnid for the saturated test item solution.

·   No males and ephippia (winter eggs) were observed in the respective control or test groups.

A summary of all test item related effects is given in Table 1. No biologically or statistically significant effects could be determined in the saturated test item solution with 1.00 mg/L test item when compared to the control.

Table 1:    Summary of all Test Item Related Effects

                   (Based on the nominal concentration of the test item of 1.00 mg/L)

		Test Item
Effects	Control	Nominal Concentration 1.00 mg/L	Significance
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	126 ± 11	126 ± 4	no
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent	9	4	no
Intrinsic Rate of Natural Increase (Mean ± SD)	0.47 ± 0.02	0.47 ± 0.02	no
Appearance of First Brood (Mean Day ± SD)	8.4 ± 0.5	8.7 ± 0.5	—
Number of Broods	5	5	—
Stillborn Juveniles and Aborted Eggs	0	3	—
Adult Mortality after 21 Days [%]	0	0	no
Parent Daphnids: Mean Dry Weight [mg]	0.93	1.10	—
Parent Daphnids: Mean Body Length [mm] ± SD	5.50 ± 0.17	5.40 ± 0.24	no
NOEC	1.00 mg/L

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

- To assure the validity of the test system and the test conditions at the test facility, an acute immobilization test according to DIN 38412 L 11 is being carried out with potassium dichromate as reference item once per month. This reference test is required by the test guideline OECD 211 (2008).
The EC₅₀ of the reference item of 1.60 mg/L (95 % confidence limits: 1.41 - 1.82 mg/L) after 24 h is 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.

Description of key information

A semi-static (daily renewal) 21-day Daphnia magna reproduction test was performed with the test item as a limit test with the saturated solution. Due to the very poor solubility of the test item, quantitative analytical determination by HPLC-DAD was precluded (below LOQ). At the saturated test item solution with 1.00 mg test item/L no biologically or statistically significant effects were determined. The No Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration (LOEC) were as follows:
NOEC : 1.00 mg/L
LOEC : > 1.00 mg/L
All provided effect values are based on the nominal concentration of the test item.