2,20-dichloro-13,31-diethyl-4,22-dioxa-13,18,31,36-tetraazanonacyclo[19.15.0.03,19.05,17.06,14.07,12.023,25.024,32.025,30]hexatriaconta-1(36),2,5,7,9,11,14,16,18,20,23,25,27,29,32,34-hexadecaene

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study according to OECD Guideline 211 and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

- Concentrations: 2.50 mg/L stock solution
No analytical determinations were carried out, because no chromatography compatible solvent is suitable for dissolving the test item in a defined and reproducible concentration

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution (2.50 mg/L test item was weighed out) was prepared with dilution water two days prior to application
- Eluate: Dilution water
- 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 [mg/L] as CaCO3
Nominal Loading
of the Test Item
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
February 18th February 20th February 25th February 27th March 4th March 6th
2.50 182 164 176 174 166 173
Control 174 165 164 163 178 182

Test temperature:

Temperature [°C]
Nominal Loading
of the Test Item
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
February 18th February 20th February 25th February 27th March 4th March 6th
2.50 21.5 20.5 21.7 21.8 21.4 21.1
Control 21.0 20.6 22.0 21.3 21.7 21.3

pH:

Nominal Loading
of the Test Item
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
February 18th February 20th February 25th February 27th March 4th March 6th
2.50 7.63 7.94 7.82 7.96 7.36 7.48
Control 7.75 7.97 7.74 8.06 7.92 7.46

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal Loading
of the Test Item
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
February 18th February 20th February 25th February 27th March 4th March 6th
2.50 8.09 9.76 8.30 8.81 7.71 8.25
Control 8.13 9.87 8.28 9.16 8.18 8.09

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Nominal: 2.50 mg/L

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 > 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
- Test concentrations: No range-finding study

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality of parent animals: All parent animals at the control group and the limit loading level survived until the end of the test. There was also no immobilization of the parent animals at the limit loading level and the control determined
- No. of offspring produced per day per female: The mean number of offspring alive produced per parent animal surviving at the end of the test was 106 juveniles at the control group and 102 juveniles at the limit loading level. The reduction of the reproductive output was calculated to be 4 %. There was no statistically significant reduction of the reproductive output (unpaired t-test, p = 0.05,
- Body length and weight of parent animals: please refer to the respective table
- Type and number of morphological abnormalities: No abnormalities
- Type and number of behavioural abnormalities: No abnormalities
- 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) = 1.53 (CI 1.43 - 1.65)

Reported statistics and error estimates:

The NOEL and LOEL were determined directly from the results.
Significant deviations were determined in comparison to the control group using statistical standard procedures as Normality Test, Equal Variance Test, Analysis of Variance and t-test.
Unpaired t-test, p = 0.05 was used for the determination of significant deviations for the reproduction rates, the intrinsic rates of natural increase and the body length. Statistical evaluation of the age of first reproduction and the occurrence of stillborn juveniles and aborted eggs could not be carried out because normality test failed. The coefficients of variation around the mean number of living offspring produced per parent in the control group and the limit loading level were evaluated.

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

Nominal Loading of the Test Item [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.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
2.50	110	96	94	98	68	104	104	96	121	129	1020	10	102 ±	17	16	4
Control	106	107	98	107	99	101	109	91	121	122	1061	10	106 ±	10	9	—

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

                        (n = 10)

Nominal Loading of the Test Item [mg/L]	Immobilization / Mortality [%]
	7 days	14 days	21 days
2.50	0	0	0
Control	0	0	0

Stillborn Juveniles and Aborted Eggs after 21 Days

Nominal Loading of the Test Item [mg/L]	Number of		Total No.		Mean Stillborn Juveniles and Aborted Eggs per Parent alive
	Stillborn	Aborted
	Juveniles	Eggs	å	N	MV ± SD
2.50	0	6	6	10	0.60 ± 1.07
Control	0	0	0	10	0 ± 0

First Appearance of Living Juveniles in the Individual Groups

Nominal Loading of the Test Item [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
2.50	8	8	8	8	8	8	8	8	8	8	8.0
Control	9	8	8	8	8	8	9	8	8	8	8.2

Total Length and Dry Weight of the Parent Animals

Nominal Loading of the Test Item [mg/L]	Total Length of the Parent Animals [mm]										MV	N	Dry Weight [mg]
	Replicate No.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
2.50	5.00	4.50	4.25	4.75	4.50	5.25	5.00	5.25	5.00	5.00	4.85	10	6.00	0.60
Control	5.00	4.75	5.00	5.00	5.25	5.00	5.00	5.00	5.00	5.25	5.03	10	5.60	0.56

Validity criteria fulfilled:

yes

Conclusions:

At the supersaturated solution of the test item with a nominal loading of 2.50 mg/L no significant effects were determined. The No Observed Effect Loading Level (NOEL) and the Lowest Observed Effect Loading Level (LOEL) were assessed at:
NOEL: 2.50 mg/L
LOEL: > 2.50 mg/L
All effect values are given based on the nominal loading level of 2.50 mg Pigment Violet 23/L of the supersaturated solution of the test item.

Executive summary:

The Daphnia magna Reproduction Test (Semi-Static, 21 d) of the test item(batch number: DEB 2060256) was conducted as a limit test with a supersaturated solution of the test item with the nominal loading of 2.50 mg/L according to OECD 211 (1998) from 2009-02-18 to 2009-03-11 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 loading level and the control. At test start they were 2 to 24 hours 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 possible, because the test item is insoluble in water and organic solvents. Therefore, the results given are based on the nominal loading of 2.50 mg test item/L.

·   The average number of juveniles per parentat the control group was 106 and 102 at the limit loading level after 21 days. There was no statistically significant reduction of the reproductive output (unpaired t-test).

·   The coefficient of variation of the mean number of living offspring produced per parent at the control group was 9 % and 16 % at the limit loading level.

·   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 mean IR of the surviving daphnids of the limit loading level was comparable to the control group (unpaired t-test, p = 0.05).

·   No immobilization or adult mortality of parent animals occurred at the control group and at the limit loading level.

·   No stillborn juveniles and aborted eggs were produced by the control group. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was 1 % at the limit loading level.

·   The first brood occurred between day 8 and 9 at each parent animal of the control and the limit loading level. In the control and test group 5 broods were released during the test period by all daphnids.

·   The mean dry body weight of the parent daphnids was 0.56 mg per daphnid at the control group and 0.60 mg per daphnid at the limit loading level. The total length of the parent daphnids of the test group was comparable to the control. The mean value of the total body length at the limit loading level was 4.85 mm per daphnid and 5.03 mm per daphnid at the control group.

·   No males and ephippia (winter eggs) occurred in control or test groups.

A summary of all test item related effects is presented in Table 1. There was no statistically significant effect determined at the supersaturated solution of the test item with a nominal loading of 2.50 mg/L.

Table 1:   Summary of all Test Item Related Effects

Based on the limit loading level of nominal 2.50 mg test item/L

Effects		Test item	Significance
	Control	Nominal Loading of the supersaturated solution 2.50 mg/L
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	106 ± 10	102 ± 17	no^1)
Intrinsic Rate of Natural Increase	0.47	0.47	no^1)
Appearance of First Brood [Mean Day]	8.2	8.0	no
Number of Broods	5	5	no
Stillborn Juveniles and Aborted Eggs	0	6	no
Adult Mortality after 21 Days [%]	0	0	no
Parent Animals: Mean Dry Weight [mg]	0.56	0.60	no
Parent Animals: Mean Body Length [mm]	5.03	4.85	no^1)
NOELReproduction	2.50 mg/L
LOELReproduction	> 2.50 mg/L

SD    = Standard deviation

no     = No biological significance

¹⁾       = Statistical significance (unpaired t-test, p = 0.05)

·   Water quality parameters as 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, determined to be 1.53 mg/L after 24 hours, 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 was also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Description of key information

At the supersaturated solution of the test item (Pigment violet 23) with a nominal loading of 2.50 mg/L no significant effects were determined. The No Observed Effect Loading Level (NOEL) and the Lowest Observed Effect Loading Level (LOEL) were assessed at: 
NOEL:      2.50 mg/L
LOEL:   > 2.50 mg/L
All effect values are given based on the nominal loading level of 2.50 mg Pigment Violet 23/L of the supersaturated solution of the test item.

Additional information

TheDaphnia magna Reproduction Test (Semi-Static, 21 d) of the test item was conducted as a limit test with a supersaturated solution of the test item with the nominal loading of 2.50 mg/Laccording to OECD 211 (1998). The test methodwas 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.

At the supersaturated solution of the test item with a nominal loading of 2.50 mg/L no significant effects were determined. The No Observed Effect Loading Level (NOEL) and the Lowest Observed Effect Loading Level (LOEL) were assessed at: NOEL: 2.50 mg/L LOEL: > 2.50 mg/L