1,4-bis(isopropylamino)anthraquinone

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 MAY 2019 to 14 JUNE 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Commission Regulation (EC) No. 440/2008, Method C.2. “Daphnia sp. Acute Immobilisation Test”, adopted 30. May 2008

Deviations:

yes

Remarks:

The temperature in the experiment was in the range 19.7 – 23.0 °C and therefore fluctuates by more than ± 1°C. As no immobility was observed in the blank control this was stated as uncritical

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 202, adopted 13. Apr. 2004:
”Daphnia sp., Acute Immobilisation Test“

Deviations:

yes

Remarks:

The temperature in the experiment was in the range 19.7 – 23.0 °C and therefore fluctuates by more than ± 1°C. As no immobility was observed in the blank control this was stated as uncritical

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis(isopropylamino)anthraquinone

EC Number:

238-101-9

EC Name:

1,4-bis(isopropylamino)anthraquinone

Cas Number:

14233-37-5

Molecular formula:

C20H22N2O2

IUPAC Name:

1,4-bis[(propan-2-yl)amino]-9,10-dihydroanthracene-9,10-dione

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1 µg/L

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: dissolution of test item.
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
- Other relevant information:

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Berlin
- Justification for species other than prescribed by test guideline:
- Age at study initiation (mean and range, SD): between 0 and 24 hours
- Weight at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD):
- Stage and instar at study initiation:
- Valve height at study initiation, for shell deposition study (mean and range, SD):
- Peripheral shell growth removed prior to test initiation:
- Method of breeding: In-house breeding since 27. September 2007
- Source: Umweltbundesamt Berlin
- Age of parental stock (mean and range, SD):
- Feeding during test: green algae (Desmodesmus subspicatus)
- Food type: green algae (Desmodesmus subspicatus)
- Amount:
- Frequency:

ACCLIMATION
- Acclimation period: 0.5 hours
- Acclimation conditions (same as test or not): yes
- Type and amount of food: green algae (Desmodesmus subspicatus)
- Feeding frequency:
- Health during acclimation (any mortality observed): no

QUARANTINE (wild caught)
- Duration:
- Health/mortality:

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
18 hours and 30 minutes before the start of the test, the adult animals were separated
from the young. 0.5 hours before test start, the adults were caught with the help of a glass
tube, and the newborn daphnia (age < 24h) were sieved from the medium and immediately placed into a beaker containing dilution water. After the settling-in period, animals which
showed no apparent damage were used for the test.
Switching from M4-medium (husbandry) to Dilution water (test) has been shown not to
cause any detrimental effects for test daphnia, therefore no acclimation period of 48 h was
performed. This is a deviation from OECD Guideline No. 202 and EU-Method C.2, which
can be considered uncritical.

Study design

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

After 24 and 48 hours, the immobilised daphnia were counted.

Test conditions

Hardness:

250 mg CaCO3/L

Test temperature:

19.7 – 23.0°C

pH:

7,6 (0h)
7.7 (48h)

Dissolved oxygen:

8.9 mg/L (0h)
8.6 mg/L (48h)

Nominal and measured concentrations:

1 µg/L (nominal concentration)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers, nominal volume 50 mL, tall shape
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass beakers, nominal volume 50 mL, tall shape
- Volume of solution: 50 ml
- Aeration: after preparation
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates):4
- No. of vessels per vehicle control (replicates):4
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: prepared water
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity: pH= 7.7
- Ca/mg ratio:
- Conductivity:
- Salinity:
- Culture medium different from test medium: M4-Medium (recipe of ELENDT)
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH: 7.7
- Photoperiod:
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
For the test concentration and the blank and solvent control, 20 daphnia were exposed to
the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised
daphnia were counted.
None of the animals was immobilised in the treatment or the blank / solvent control. There
fore no statistical evaluation was performed.
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a
current reference study to assure that the test conditions are reliable.
VEHICLE CONTROL PERFORMED: No

RANGE-FINDING STUDY
- Test concentrations: 1 µg/L
- Results used to determine the conditions for the definitive study: 1 µg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study (201902R201). The 24h-EC50 value was determined as 1.98 mg/L. The value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L of po

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: For the test concentration and the blank and solvent control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted. None of the animals was immobilised in the treatment

Effect concentrations 2

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: For the test concentration and the blank and solvent control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted. None of the animals was immobilised in the treatment

Effect concentrations 3

Key result

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

> 1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: For the test concentration and the blank and solvent control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted. None of the animals was immobilised in the treatment

Effect concentrations 4

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: For the test concentration and the blank and solvent control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted. None of the animals was immobilised in the treatment

Effect concentrations 5

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.79 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: For the test concentration and the blank and solvent control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted. None of the animals was immobilised in the treatment

Effect concentrations 6

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 0.79 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: For the test concentration and the blank and solvent control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted. None of the animals was immobilised in the treatment

Effect concentrations 7

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 0.79 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: For the test concentration and the blank and solvent control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted. None of the animals was immobilised in the treatment

Effect concentrations 8

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.79 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: For the test concentration and the blank and solvent control, 20 daphnia were exposed to the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted. None of the animals was immobilised in the treatment

Details on results:

- Behavioural abnormalities: no
- Observations on body length and weight: no
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control:no
- Immobilisation of control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels:
- Limit test:
- Dose-response test:
- ECx:
- Other: Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a
current reference study to assure that the test conditions are reliable.

Reported statistics and error estimates:

None of the animals was immobilised in the treatment or the blank / solvent control. There
fore no statistical evaluation was performed.

Any other information on results incl. tables

Table 9.1-a Biological Results Test Item

Parameter	Based on nominal values	Based on measured values
24h EC50	> 1 µg/L	> 0.79 µg/L
48h EC50	> 1 µg/L	> 0.79 µg/L
48h NOEC	≥ 1 µg/L	≥ 0.79 µg/L
48h LOEC	> 1 µg/L	> 0.79 µg

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

One valid experiment was performed.
The solubility of the test item in test medium is < 1 µg/L (see validation report
AB18062904G926). Therefore, this study was performed as a limit test at 1 µg/L to show
that there is no toxicity at the proposed LOQ (limit of quantification) of the analytical method. Acetone was used to spike the test solution. A stock solution containing 10 mg/L in
acetone were prepared. The concentration of the stock solution is 1000-fold higher than
the nominal concentration in the treatment (1 µg/L). For the solvent control, acetone in a
concentration of 100 µL/L was used.
For the test concentration and the blank and solvent control, 20 daphnia were exposed to
the test item for 48 hours in a static test system. After 24 and 48 hours, the immobilised
daphnia were counted.
None of the animals was immobilised in the treatment or the blank / solvent control. Therefore no statistical evaluation was performed.
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a
current reference study to assure that the test conditions are reliable.
At the beginning and at the end of the test, the content of the test item in the test solutions
was determined using LC/MS-MS-determination. All measured values are below the LOQ
(1 µg/L). At the beginning of the test, however, the measured raw data (area) clearly indicate that the test item is present in the solution. At the end of the test no test item was detectable. Due to the very low concentration, it is not clear what caused the loss of substance (hydrolysis, precipitation, or adsorption to the test organisms).
Therefore, the measured initial value is used for evaluation, even if it is below the LOQ.
In addition, the results are displayed in relation to the nominal values. This is in agreement
with the OECD guidance document no. 23 chapter 9: “For tests with chemicals that cannot
be quantified by the most sensitive analytical methods at relevant concentrations, the effect concentration can be expressed based on the nominal concentrations”.
No observations were made which might cause doubts concerning the validity of the study
outcome. All validity criteria were met.
The result of the test is considered valid