2,2'-[ethylenebis(oxyphenyl-2,1-eneazo)]bis[N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-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:

experimental study

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: Study 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 with a nominal loading of 1 mg/L as limit concentration.
- Sampling method: Analytical evaluation of the saturated solution of the 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. The nominal test item concentration was used for estimation of the endpoints.
- 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: Stock solutions of 1 mg/L were prepared with dilution water two days prior to application / water renewal. The stock solution was stirred with approx. 1100 rpm for 48 h at room temperature. Undissolved particles were removed by membrane filtration.
- Eluate: Dilution water
- Differential loading: None, limit test
- 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:

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) 173 164 160 166 176 164
Control 176 162 176 169 176 164

Test temperature:

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) 21.6 20.7 21.3 21.6 20.6 21.3
Control 20.1 20.6 20.3 21.5 20.8 21.1

pH:

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) 8.15 7.94 7.65 7.54 7.86 7.42
Control 7.92 7.80 7.79 7.49 7.95 7.46

Dissolved oxygen:

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) 6.78 8.90 6.58 8.44 6.18 7.55
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
A preliminary test was carried out as acute immobilization test with the saturated solution with 1 mg test item/L under static conditions over a period of 48 hours. No effects (including immobilization, signs of disease or stress, e.g. discoloration or unusual behaviour) were observed on Daphnia magna after an exposure period of 48 hours.
Immobilization Rates in the Preliminary Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item Concentration

[mg] 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 was precluded (below LOQ)

Details on results:

- Mortality of parent animals:
No adult mortality or immobilization of parental daphnids was observed in the 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 test 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 parental daphnids in the control and test groups. The mean number of alive offspring produced per parent daphnid surviving the test was 126 in the control and 121 in the saturated test item solution with 1.00 mg test item/L after 21 days, respectively. The reproductive output of daphnids in the saturated test item solution (limit concentration) was comparable to the output of daphnids in the control (t-test, p = 0.05).

- Body length and weight of parental animals:
Please refer to the respective table

- Type and number of morphological / behavioural abnormalities:
No stillborn juveniles and aborted eggs were observed in the control and the saturated solution with 1.00 mg test item/L during the exposure period of 21 days. No ephippia were observed in any group during the test.

- 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, the intrinsic rates of natural increase and the total 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.
The EC50-value was calculated for the reference dose-response test by sigmoidal dose-response regression. The confidence interval for the EC50-value was calculated from the best-fit dose-response curve, standard error and t-distribution using the software GraphPad prism.

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)	120	102	118	126	118	118	134	126	133	115	1210	10	121 ±  9	8		4		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

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)	9	8	9	9	9	8	8	9	9	8	8.6
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]	Stat.		Sum	MV
1.00 (Saturated solution)	5.75	5.50	5.50	5.25	5.75	5.00	5.50	5.25	5.50	5.50	5.45	no	10	10.3	1.03
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

Stat. = statistically significant difference

no = no statsitically significant difference

Validity criteria fulfilled:

yes

Conclusions:

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, 21 d) of 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).

The measured concentrations of the test item were below the limit of quantification of the analytical method (LOQ_M) 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 121 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 (t-test, p = 0.05).

·   The coefficient of variation of the number of living offspring produced per parent was 9 % for the control and 8 % for the saturated test item solution*.

·   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* (Table 5).

·   No dead juveniles and aborted eggs were observed in control and the saturated solution*.

*)    Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6. 

·   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 1.03 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 5.45 mm per daphnid for the saturated test item solution. The total body lengths of all daphnids in the saturated test item solution were comparable to the total body lengths in the control (t-test, p = 0.05).

·   No malesand 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 effect could be determined in the saturated test item solution with 1.00 mg/L test item when compared to the control.

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	121 ± 9	no^1)
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent	9	8	no
Intrinsic Rate of Natural Increase (Mean ± SD)	0.47 ± 0.02	0.46 ± 0.02	no^1)
Appearance of First Brood (Mean Day ± SD)	8.4 ± 0.5	8.6 ± 0.5	—
Number of Broods	5	5	—
Stillborn Juveniles and Aborted Eggs	0	0	—
Adult Mortality after 21 Days [%]	0	0	no
Parent Daphnids: Mean Dry Weight [mg]	0.93	1.03	—
Parent Daphnids: Mean Body Length [mm] ± SD	5.50 ± 0.17	5.45 ± 0.23	no^1)
NOEC	1.00 mg/L

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-11-14 to 2012-01-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study 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: Analytical evaluation of the saturated solution of the 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.
- Sampling method: For the old media, separate replicates without test organisms were prepared and incubated under test conditions. The nominal test item concentration was used for estimation of the endpoints.
- 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: A stock solution of 1 mg/L was prepared with dilution water 2 days prior to application/water renewal.The stock solution was stirred with approximately 1100 rpm for 48 hours at room temperature. Undissolved particles were removed by membrane filtration (membrane filter: 0.45 µm, RC, MACHEREY-NAGEL). Due to the test item properties centrifugation was not possible.
- Eluate: Dilution water
- Differential loading: None, limit test
- 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) 160 160 174 164 176 164
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 20.6 21.1 21.6 20.5 21.1
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) 8.27 7.79 7.61 7.52 7.70 7.53
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) 5.99 8.31 6.72 8.32 6.37 8.66
Control 8.84 8.53 8.76 7.49 8.85 7.34

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Due to the extremely low water solubility of the substance it was not possible to detect the substance in the treatments (below LOQ).

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 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] 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 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 test 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 123 in the saturated test item solution with 1.00 mg test item/L after 21 days, respectively. The reproductive output of daphnids in the saturated test item solution (limit concentration) was comparable to the output of daphnids in the control (t-test, p = 0.05).
- Body length and weight of parent animals:
Please refer to the respective table

- 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. No ephippia were observed in any group during the test.
- 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 and the intrinsic rates of natural increase 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)	128	120	121	103	117	121	136	144	126	118	1234	10	123 ± 11	9		2		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	2	1234	1236	1
Control	0	1260	1260	0

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	8	9	9	8	9	9	8	9	8.5
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)	5.25	5.25	5.25	5.50	5.50	5.25	5.75	5.75	5.50	5.50	5.45	10	10.1	1.01
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

Validity criteria fulfilled:

yes

Conclusions:

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, 21 d) of 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).

The measured concentrations of the test item were below the limit of quantification of the analytical method (LOQ_M) 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 123 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 (t-test, p = 0.05).

·   The coefficient of variation of the number of living offspring produced per parent was 9 % for the control and 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 1.01 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 5.45 mm per daphnid for the saturated test item solution.

·   No malesand 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 effect 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	123 ± 11	no^1)
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent	9	9	no
Intrinsic Rate of Natural Increase (Mean ± SD)	0.47 ± 0.02	0.46 ± 0.02	no^1)
Appearance of First Brood (Mean Day ± SD)	8.4 ± 0.5	8.5 ± 0.5	—
Number of Broods	5	5	—
Stillborn Juveniles and Aborted Eggs	0	2	—
Adult Mortality after 21 Days [%]	0	0	no
Parent Daphnids: Mean Dry Weight [mg]	0.93	1.01	—
Parent Daphnids: Mean Body Length [mm] ± SD	5.50 ± 0.17	5.45 ± 0.20	—
NOEC	1.00 mg/L

SD = Standard deviation

no = no biological significance

1) = no statistical significance

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 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

Justification for type of information:

see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Acetolone Pigments – Nanoforms and Bulk Forms (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other:

Remarks:

ead across from PY154 and PO36

Validity criteria fulfilled:

yes

Conclusions:

At the saturated test item solution (P036) 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

At the saturated test item solution (PY154) 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
Due to the very poor solubility of the test item, quantitative analytical determination was precluded (below LOQ)

Description of key information

At the saturated test item solution (P036 bulk) 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

 

At the saturated test item solution (PY154 bulk) 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

Due to the very poor solubility of the test item, quantitative analytical determination was precluded (below LOQ)