Reaction mass of isomers 4-[[2-[(Aminocarbonyl)amino]-4-[(2,6-difluoro-4-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt and 4-[[2-[(Aminocarbonyl)amino]-4-[(4,6-difluoro-2-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-06-28 to 2013-07-22

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

148559-52-8 (as free acid)

IUPAC Name:

148559-52-8 (as free acid)

Details on test material:

- Name of test material (as cited in study report): Reactive Golden Yellow HF-RN 1331
- Molecular formula: C17H11F2Na2O7S2 (as sodium salt) C17H13F2N7O7S2 (as free acid)
- Physical state: Solid, red to orange powder
- Analytical purity: 100 %
- Purity test date: 2012-10-08
- Lot/batch No.: Kilo 10 & 12
- Expiration date of the lot/batch: 2014-01-17
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature, protected from light, in original container; since 2013-03-01 additionally under nitrogen

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Limit test with 100 mg/L.

- Sampling method: The limit concentration of 100 mg/L of the test item and the control were analysed by HPLC-DAD at the start of the exposure intervals (0 h) on days 0, 10, 17 and at the end of the exposure intervals (48 or 72 h) on days 3, 12, 19.
For the analyses at the end of the exposure intervals, separate replicates without algae and daphnids were prepared and stored under test conditions until sampling.


- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C. Prepared samples were stored at room temperature (autosampler) until analysis, if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Agitation. The limit concentration (100 mg/L test item were weighed out) was freshly prepared with dilution water at the start of each exposure interval at every water renewal day.


- Eluate: Dilution water
- Differential loading: 100 mg/L
- Controls: 10 replicates of dilution water without test item

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Frequency: renewals 3 times per week


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

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

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Test conditions

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
2013-06-28 2013-07-01 2013-07-08 2013-07-10 2013-07-15 2013-07-17
100 180 169 160 178 164 150
Control 178 142 174 164 174 190

Test temperature:

Temperature [°C]
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
2013-06-28 2013-07-01 2013-07-08 2013-07-10 2013-07-15 2013-07-17
100 20.8 21.5 21.2 21.5 22.0 21.6
Control 20.6 21.6 21.5 21.7 21.9 21.6

pH:

pH-Values
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
2013-06-28 2013-07-01 2013-07-08 2013-07-10 2013-07-15 2013-07-17
100 7.84 8.21 7.85 7.64 7.69 7.55
Control 7.75 8.22 8.42 7.69 7.66 7.59

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
2013-06-28 2013-07-01 2013-07-08 2013-07-10 2013-07-15 2013-07-17
100 8.66 9.09 8.47 7.58 7.83 7.41
Control 8.48 9.37 8.20 8.04 8.21 7.56

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

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: on days 9 and 10
- Intrinsic rate of natural increase: test end
- Growth (total length and dry weight): test end


RANGE-FINDING STUDY: None

Based on the results of an acute immobilization test conducted at the test facility (Lab-ID: 130215CA/DAI15440) under static conditions over a period of 48 hours, the definitive reproduction test was carried out as a limit test with the limit concentration of 100 mg/L of the test item Reactive Golden Yellow HF-RN 1331.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality of parent animals:
No adult mortality was observed during the test period of 21 days. In the limit concentration of 100 mg/L and in the control, all parental daphnids survived until the end of the test after 21 days.
- No. of offspring produced per day per female:
No stillborn juveniles or aborted egg were observed in the control. At the limit concentration of 100 mg/L, two aborted eggs were observed during the exposure period of 21 days, which corresponds to 0.2 % and is therefore negligible.
- Body length and weight of parent animals:
The mean dry body weight and total body lengths of all parental daphnids of the limit concentration and the control were determined at the end of the study. The mean dry body weight of the parental daphnids was 0.97 mg per daphnid for the control and 0.83 mg per daphnid for the limit concentration of 100 mg/L.
The mean body length of the parental daphnids was 5.08 mm per daphnid for the control and 5.30 mm per daphnid for the limit concentration.

- Type and number of morphological / behavioural abnormalities: please refer to the respective table
- Time to first brood release or time to hatch:
The first brood was released on days 9 and 10 by all parental daphnids of the control and the limit concentration of 100 mg/L.
At the limit concentration of 100 mg/L and the control, 4 broods were produced by all parental daphnids.
- Presence of males: No males were observed in the control or in the test groups during the test.
- Occurrence of Ephippia (Winter Eggs): No ephippia were observed in the control or in the test groups during the test.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.92 (CI 1.74 - 2.11) mg/L

Reported statistics and error estimates:

The NOEC for the reproduction as well as adult survival was deduced directly from the results.
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 a t-test, a normality test and an equal variance test were performed. P-values of the normality and equal variance tests were set to 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) was =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 living offspring produced per parent were calculated for the control and the limit concentration.

Any other information on results incl. tables

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

Nominal test item conc. [mg/L]												No. of parents prod. juv.	Mean no. of juveniles per parent producing juveniles			Comparison versus control
	Number of living juveniles in replicate no.										Total no.				CV
																Inc.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
100	91	85	83	83	94	96	75	85	91	80	863	10	86 ±	7	8	3	no
Control	93	80	86	84	85	75	82	77	89	84	835	10	84 ±	5	6	—	—

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

                 (n = 10)

Nominal Test Item Concentration [mg/L]	Adult Mortality [%]
	7 days	14 days	21 days
100	0	0	0
Control	0	0	0

First Appearance of Living Juveniles and Number of Broods in the Individual Groups

Nominal test item concentration [mg/L]	Day of first appearance of living juveniles										First	Mean
	in replicate no.										appearance	number
	1	2	3	4	5	6	7	8	9	10	mean day	of broods
100	9	9	10	9	9	9	9	10	9	9	9.2	4
Control	9	9	9	9	9	9	10	9	9	9	9.1	4

Total Body Length and Dry Weight of the Parental Daphnids

Nominal test item concentration [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
100	5.25	5.50	5.00	5.50	5.50	5.50	5.25	5.00	5.25	5.25	5.30	10	8.3	0.83
Control	5.25	5.00	4.75	5.00	5.25	5.25	5.00	5.00	5.00	5.25	5.08	10	9.7	0.97

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

At the limit concentration of 100 mg/L of the test item Reactive Golden Yellow HF-RN 1331, no biologically or statistically significant effects were determined. Based on the nominal concentration, the No Observed Effect Concentration (NOEC) was 100 mg/L.

Executive summary:

The Daphnia magna Reproduction Test (Semi-Static, 21 d) was conducted as a limit test with the limit concentration of 100 mg/L of the test item Reactive Golden Yellow HF-RN 1331 (batch number: Kilo 10&12) according to OECD 211 (2008) from 2013-06-28 to  2013-07-22, with the definitive exposure phase from 2013-06-28 to 2013-07-19, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany.

The test species was Daphnia magna STRAUS (Clone 5). Ten daphnids individually held were used for the limit concentration and the control. At the start of the exposure, the daphnids were 2 to 24 h old. The test methodwas semi-static with renewal of the test solutions three times per week. The 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 intrinsic rate of natural increase, occurrence of aborted eggs and stillborn juveniles, time of production of the first brood, adult mortality, dry body weight and length of the parental daphnids.

The limit concentration of the test item Reactive Golden Yellow HF-RN 1331 was analytically verified by HPLC-DAD of samples taken at the start of the exposure intervals on days 0, 10, 17 (0 h) and at the end of the exposure intervals on days 3, 12, 19 (48 or 72 h) of the limit concentration and the control.

The recovery of the limit concentration of the test item at the start of the exposure intervals was in the range of 99 to 102 % of the nominal concentration of 100 mg/L. At the end of the exposure intervals (48 or 72 h), the recovery of the limit concentration of the test item was in the range of 101 to 106 % of the nominal concentration. This demonstrates, that the test item concentration was successfully maintained for the duration of the test. The results given are based on the nominal concentration of 100 mg/L of the test item Reactive Golden Yellow HF-RN 1331.

·   The average values of living juveniles per parental daphnidwere 84 for the control and 86 for the limit concentration of 100 mg/L after 21 days. The reproductive output of daphnids in the limit concentration was comparable to the reproductive 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 6 % for the control and 8 % for the limit concentration of 100 mg/L.

·   The intrinsic rates of natural increase (IR) of the surviving parental daphnids accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The intrinsic rates of the limit concentration were comparable to those in the control (t-test, p = 0.05).

·   No adult mortality was observed during the test period of 21 days. In the limit concentration of 100 mg/L and in the control, all parental daphnids survived until the end of the test after 21 days.

·   The first brood was released on days 9 and 10 by all parental daphnids of the control and the limit concentration of 100 mg/L.
At the limit concentration of 100 mg/L and the control, 4 broods were produced by all parental daphnids.

·   The mean dry body weight and total body lengths of all parental daphnids of the limit concentration and the control were determined at the end of the study.
The mean dry body weight of the parental daphnids was 0.97 mg per daphnid for the control and 0.83 mg per daphnid for the limit concentration of 100 mg/L.
The mean body length of the parental daphnids was 5.08 mm per daphnid for the control and 5.30 mm per daphnid for the limit concentration.

·   No stillborn juveniles or aborted eggs were observed in the control. At the limit concentration of 100 mg/L, two aborted eggs were observed during the exposure period of 21 days, which corresponds to 0.2 % and is therefore negligible.

·       No males or ephippia (winter eggs) were observed in the control or in the limit concentration of 100 mg/L.

 

·   Water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and temperature) were within the acceptable limits.

No biologically significant effect could be determined in the limit concentration of 100 mg/L in comparison to the control.

         

Summary of all Test Item Related Effects

(Based on the nominal test item concentration of 100 mg/L)

Reactive Golden Yellow HF-RN 1331
Effects		Control	Nominal test item concentration 100 mg/L	Significant effect
Mean number of juveniles per producing parent (Reproduction rate ± SD)		84 ± 5	86 ± 7	no^1)
Coefficient of variation of the mean number of juveniles per producing parent		6	8	—
Intrinsic rate of natural increase (Mean ± SD)		0.42 ± 0.02	0.42 ± 0.02	no^1)
Number of stillborn juveniles and aborted eggs		0	2	—
Appearance of first brood (Mean day ± SD)		9.1 ± 0.3	9.2 ± 0.4	—
Number of broods		4	4	—
Adult mortality after 21 days [%]		0	0	no
Parental daphnids:	Mean dry weight [mg]	0.97	0.83	—
	Mean body length [mm] ± SD	5.08 ± 0.17	5.30 ± 0.20	—
NOEC		100 mg/L

In order to prove the validity of the test system and test conditions at the test facility, an acute immobilisation test according to DIN 38412 L 11 is monthly carried out with potassium dichromate as reference item.
The EC₅₀ of the reference item was 1.92 mg/L (95% confidence limits: 1.74 - 2.11 mg/L) after 24 hours and is therefore within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) 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.