reaction mass of: tetrasodium 4-amino-6-(5-(2,6-difluoropyrimidin-4-ylamino)-2-sulfonatophenylazo)-5-hydroxy-3-(4-(sulfatoethylsulfonyl)phenylazo)naphthalene-2,7-disulfonate;tetrasodium 4-amino-6-(5-(4,6-difluoropyrimidin-2-ylamino)-2-sulfonatophenylazo)-5-hydroxy-3-(4-(2-sulfatoethylsulfonyl)phenylazo)naphthalene-2,7-disulfonate

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

Study period:

21 March 2001 to 11 April 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted in accordance with the OECD test guideline 211 (Daphnia magna reproduction test) in compliance with GLP. However, only three test concentrations were employed (guideline requirement: 5), the test concentrations were too low for identifying an NOEC and LOEC, and the measured test substance concentration fluctuated between 84 and 196 % of nominal over the 21 duration of the study. This does not invalidate the study, but only limits its reliability. The concentration fluctuations were addressed during preparation of this study summary by calculating the mean measured concentration.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1, 3.2 and 10 mg/L and blank control
- Sampling method: No data
- Sampling frequency: Stock solution - days 0, 2, 5, 7, 9, 12, 14, 16 and 19
- Sampling frequency: Control - days 0, 7, 16 in new media and 2, 9, 19 in old media
- Sampling frequency: test concentrations - days 0, 7, 16 in new media and 2, 9, 19 in old media
- Sample storage conditions before analysis: No data

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

Test organism:
- Common name: not stated
- Strain: Bundesgesundheitsamt Berlin
- Maintenance and acclimation: a population of parthenogenetic females of synchronised age structure were maintained for more than 15 years in the test facility.
- Weight at study initiation (mean and range, SD): No data
- Method of breeding: In M4 medium with weekly partly renewal, at 20 ± 1 °C, 16:8 h light/dark cycle, feeding with unicellular algae (Scenedesmus subspicatus CHODAT) ad libitum.
- Feeding during test: Scenedesmus subpicatus CHODAT, day 1-7: 0.1 mg/C/Daphnia/day and day 8-21: 0.2 mg C/Daphnia/day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not conducted

Hardness:

Overall the hardness of the dilution water ranged from 14.9 -15.9 °dH (=266-283.8 mg/L CaCO3) during the test
Measured hardness for control: 14.9-15.8 °dH
Measured hardness for 1 mg/L nominal concentration: not stated
Measured hardness for 3.2 mg/L nominal concentration: not stated
Measured hardness for 10 mg/L nominal concentration: 14.9-15.9 °dH

Test temperature:

Nominal: 18-22 °C, mean ± 1 °C
Measured temperature for control: 18.5-20.2 °C
Measured temperature for 1 mg/L nominal concentration: not stated
Measured temperature for 3.2 mg/L nominal concentration: not stated
Measured temperature for 10 mg/L nominal concentration: 18.6 -20.3 °C

pH:

Measured pH for control: 7.6-7.9
Measured pH for 1 mg/L nominal concentration: not stated
Measured pH for 3.2 mg/L nominal concentration: not stated
Measured pH for 10 mg/L nominal concentration: 7.6-7.9

Dissolved oxygen:

Measured oxygen concentration (saturation) for control: 7.5-9.3 mg/L (84.7-102.2 %)
Measured oxygen concentration (saturation) for 1 mg/L nominal concentration: not stated
Measured oxygen concentration (saturation) for 3.2 mg/L nominal concentration: not stated
Measured oxygen concentration (saturation) for 10 mg/L nominal concentration: 7.5-9.1 mg/L (84.4- 100.8%)

Salinity:

No stated

Nominal and measured concentrations:

Nominal concentration: 1, 3.2 and 10 mg/L
Measured concentration: 8.4-19.6 mg/L (based on 1mg/L TOC is equivalent to 2.8 mg/L of test substance)
Only the 10 mg/L concentration was measured due to the limit of detection
- Intervals of water quality measurement control and test vessels: measured on day 0, 7 and 19 for old media (after renewal) and 2, 9 and 19 for new media (before renewal)

Details on test conditions:

TEST SYSTEM
- Material, size, headspace, fill volume: Glass beaker, 150 mL, containing 100 mL test medium
- Aeration: No
- Renewal rate of test solution: renewal of test media on Mondays, Wednesdays and Fridays
- 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: Reconstituted water (M4 medium)
- Culture medium different from test medium: Same media used for maintenance of test animals, preparation of stock and test solutions


OTHER TEST CONDITIONS
- Adjustment of pH: No data
- Photoperiod: 16:8 h (L/D)
- Light intensity: not exceeding 800 lux

EFFECT PARAMETERS MEASURED
- mortality and reproduction

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 14 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 14 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 14 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 14 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

-The measured concentrations of test material varied between 84 and 196 % of the nominal concentration (based on the 10 mg/L nominal concentration)
- No mortality or immobilisation of parent Daphnia was recorded.

Reported statistics and error estimates:

-Method: The NOEC (reproduction) and the NOEC (mortality) for parent Daphnia was determined using DUNNETT's test (1955, 1964), which is a multiple comparison procedure for comparing several treatments with a control.
- Result: The test showed no significant difference between the test solutions and the control.

Mean reproduction rate

Nominal concentration [mg/I]	Mean measured concentration [mg/I)	Mean reproduction rate	SD	CV [%]
Control	----	123.3 [n=10]	18.8	15.3
1	----	133.5 [n=10]	17.3	13
3.2	----	123.8 [n=10]	18.4	14.9
10	14	112 [n=l0]	19.5	17.4

Immobilisation /mortality rate and mean reproduction

Nominal concentration (mg/L)	Mean measured concentration (mg/L)	Immobilisation/mortality rate of parent Daphnia		Mean reproduction rate of parent Daphnia
		Absolute	%	Absolute	%
Control		0	0	123.3 (n=10)	100
1		0	0	133.5 (n=10)	108
3.2		0	0	123.8 (n=10)	100
10	14	0	0	112 (n=10)	91

Validity criteria fulfilled:

yes

Remarks:

The validity of the test in terms of mortality rate of controls, living offspring of parents, DO concentration, pH and water hardness were fulfilled. The test material concentration varied dramatically over the duration of the exposure.

Conclusions:

Test organisms (Daphnia magna) were exposed to nominal test substance concentrations of 0, 1, 3.2 and 10 mg/L for 21 days. The 21 day EC50 values for mortality and reproduction were both > 14mg/L (mean measured concentration; variation by 84-196 % of the nominal over the study duration). The 21 day NOEC for reproduction and mortality were both ≥ 14 mg/L.

Executive summary:

The 21-day-chronic toxicity of Reactive Navy FC 63805 to Daphnia magna was studied under semi-static conditions. Daphnids were exposed to control, and test chemical at nominal concentrations (measured concentrations) of 0, 1, 3.2, and 10 mg/L (mean measured: 14 mg/L). The 21 day EC₅₀ based on mortality and reproduction effects was > 14 mg/L. The 21 day NOECs based on mortality and reproduction were both ≥ 14mg/L. No mortality or sublethal effects were recorded. Production of offspring in the treated groups indicated that the test chemical did not have an effect on the reproduction at concentrations of at least 14 mg/L.

This study is classified as acceptable and partly satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates. The number of test concentrations was less than prescribed by the guideline, and concentrations were not sufficiently high to induce effects, so no NOEC and LOEC could be identified. The test substance concentration fluctuated between 84 and 196 % of the nominal concentration, therefore the (unbounded) NOEC is given as the mean measured concentration of 14 mg/L

Results synopsis

Test type: Static Renewal

NOEC ≥ 14 mg/L (reproduction and mortality)

LOEC > 14 mg/L (reproduction and mortality)

Description of key information

Test organisms (Daphnia magna) were exposed to nominal test substance concentrations of 0, 1, 3.2 and 10 mg/L for 21 days. The 21 day EC50 values for mortality and reproduction were both > 14 mg/L (mean measured concentration; variation by 84-196 % of the nominal over the study duration). The 21 day NOEC for reproduction and mortality were both ≥ 14 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

14 mg/L

Additional information

The 21-day-chronic toxicity of Reactive navy FC 63805 to Daphnia magna was studied under semi-static conditions. Daphnids were exposed to control, and test chemical at nominal concentrations (measured concentrations) of 0, 1, 3.2, and 10 mg/L (mean measured: 14 mg/L). The 21 day EC₅₀ based on mortality and reproduction effects was > 14 mg/L. The 21 day NOECs based on mortality and reproduction were both ≥ 14mg/L. No mortality or sublethal effects were recorded. Production of offspring in the treated groups indicated that the test chemical did not have an effect on the reproduction at concentrations of at least 14 mg/L.

This study is classified as acceptable and partly satisfies the guideline requirements for a chronic toxicity study with freshwater invertebrates. The number of test concentrations was less than prescribed by the guideline, and concentrations were not sufficiently high to induce effects, so no NOEC and LOEC could be identified. The test substance concentration fluctuated between 84 and 196 % of the nominal concentration, therefore the (unbounded) NOEC is given as the mean measured concentration of 14 mg/L.

Since the NOEC is unbounded it is not suitable as the key value for safety assessment. No relevant NOEC or EC10 for the chemical safety assessment can be derived.