Ethanaminium, N-[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthalenyl]methylene]-2,5-cyclohexadien-1-ylidene]-N-ethyl-, molybdatetungstatephosphate

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:

14 November 2017 - 10 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 278PE160900 supplied by Client
- Expiration date of the lot/batch: 01 July 2022
- Purity test date: N/a


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Solutions of the test substance im Elendt M7 medium prepared as a WAF due to low aqueous solubility and complex nature of the test substance
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/a
- Preliminary purification step (if any): N/a
- Final dilution of a dissolved solid, stock liquid or gel: WAFs prepared at a 1.0, 10 or 100 mg/L loading rate
- Final preparation of a solid:

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: Samples of the fresh test preparations were taken on Days 0, 6, 13 and 20 and of the expired test preparations on Days 1, 7, 14 and 21.
- Sample storage conditions before analysis: Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: WAF preparation
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Elendt M7 medium
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.16, 0.40, 1.0, 2.5 and 6.4 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Microscopic examination of WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). Microscopic observations of the WAFs were performed after filtering and showed no microdispersions of test item present.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: In-house laboratory cultures
- Justification for species other than prescribed by test guideline:
- Source: in-house stock
- Age of parental stock (mean and range, SD): Yound daphnids less than 24 hours old
- Feeding during test Fed daily
- Food type: algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
- Amount: not specified
- Frequency: Daily

ACCLIMATION
- Acclimation period:N/a
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food:Not speified
- Feeding frequency: daily
- Health during acclimation (any mortality observed): good


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.

Test type:

static

Water media type:

other: Elendt M7 medium

Limit test:

no

Total exposure duration:

21 d

Hardness:

244 - 264 mg/L CaCO3

Test temperature:

18 to 22 °C

pH:

7.7 - 8.1

Dissolved oxygen:

8.0 - 8.9 mg O2/L

Nominal and measured concentrations:

Nominal loading rates (mg/L): 0.16, 0.40, 1.0, 2.5 and 6.4
Measured concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass vessels
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: fill volume 100 mL
- Aeration: N/a
- Type of flow-through (e.g. peristaltic or proportional diluter): N/a
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel:1 daphnid
- No. of vessels per concentration (replicates):5
- No. of vessels per control (replicates):5
- No. of vessels per vehicle control (replicates):
- Biomass loading rate:n/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days
- Light intensity: <500 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5 for main study
- Justification for using less concentrations than requested by guideline:
- Range finding study: Yes
- Test concentrations: 1.0. 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.98 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

The LOEL was 0.40 mg/L as the body length of the parental generation daphnids in this test group were significantly smaller (P<0.05) than the daphnids in the control group.
The NOEL was 0.16 mg/L as there were no significant mortalities (immobilization) observed in the parental generation (P1), there were no significant differences (P≥0.05) in terms of the number of live young produced per adult and the body length of the parental generation daphnids in this test group were not significantly different when compared to the control after 21 days.

Validity criteria fulfilled:

yes

Conclusions:

The LOEL was 0.40 mg/L as the body length of the parental generation daphnids in this test group were significantly smaller (P<0.05) than the daphnids in the control group.
The NOEL was 0.16 mg/L as there were no significant mortalities (immobilization) observed in the parental generation (P1), there were no significant differences (P≥0.05) in terms of the number of live young produced per adult and the body length of the parental generation daphnids in this test group were not significantly different when compared to the control after 21 days.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have the same type of toxicological effects based on common underlying mechanisms (ECHA, 2015b). All substances have the same organic structural group (colorant) with different compositions of the inorganic group (metallic salt). Read-across is in this case based on the hypothesis that source and target substances have similar toxicological properties because they degrade to the similar chemical substances (ECHA, 2015c). This prediction is supported by physicochemical and toxicological data on the substances.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Name Target Substance Source Substance
Chemical name Ethanaminium, N-[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthalenyl]methylene]- Bis[[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthyl]methylene]cyclohexa-2,5-
2,5-cyclohexadien-1-ylidene]-N-ethyl-, molybdatetungstatephosphate dien-1-ylidene]diethylammonium] dicopper(1+) hexa(cyano-C)ferrate(4-)
IUPAC name Reaction product of [4-[bis[4-(diethylamino)phenyl]methylidene]naphthalen-1-ylidene]-
ethylazanium chloride with phosphotungstomolybdic acid Unknown
CAS No 1325-87-7 82338-76-9/57485-98-0
EC No 215-410-7 279-935-3
Colourant C.I. Basic Blue 7 (R30200) C.I. Basic Blue 7 (R30200)
Metals Phosphotungstomolybdic acid salt Copper ferrocyanide salt
Molecular weight range ≥ 6600 ≤ 10500 880-1750
Molecular formula (C33H40N3)z.O3.P2O5.(WO3)x.(MoO3)y C33H40N3.1/2C6FeN6.Cu
12 ≤ x+y ≤ 24 (x≥1; y≥1)
z = 6-10
Structural formula

Both substances are UVCB’s and so purity is considered to be 100%

3. ANALOGUE APPROACH JUSTIFICATION
The target substance CAS 1325-87-7is an organometallic multi-constituent substance. The source substance CAS 63022-06-0 has the same colourant (organic constituent) (see table above) as the target substance consisting of a basic [4-[bis[4-(diethylamino)phenyl]methylidene]naphthalen-1-ylidene]-ethylazanium. The inorganic Part B is different between source and target substances, consisting of the elements O, Mo, P and W for the target substance and Cu, Fe and CN for the source substance. These differences are expected to behave very similar. The target and source substances are thus considered suitable for the analogue approach based on structural similarity.

Reason / purpose for cross-reference:

read-across source

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: Samples of the fresh test preparations were taken on Days 0, 6, 13 and 20 and of the expired test preparations on Days 1, 7, 14 and 21.
- Sample storage conditions before analysis: Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: WAF preparation
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Elendt M7 medium
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.16, 0.40, 1.0, 2.5 and 6.4 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Microscopic examination of WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). Microscopic observations of the WAFs were performed after filtering and showed no microdispersions of test item present.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: In-house laboratory cultures
- Justification for species other than prescribed by test guideline:
- Source: in-house stock
- Age of parental stock (mean and range, SD): Yound daphnids less than 24 hours old
- Feeding during test Fed daily
- Food type: algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
- Amount: not specified
- Frequency: Daily

ACCLIMATION
- Acclimation period:N/a
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food:Not speified
- Feeding frequency: daily
- Health during acclimation (any mortality observed): good


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.

Test type:

static

Water media type:

other: Elendt M7 medium

Limit test:

no

Total exposure duration:

21 d

Hardness:

244 - 264 mg/L CaCO3

Test temperature:

18 to 22 °C

pH:

7.7 - 8.1

Dissolved oxygen:

8.0 - 8.9 mg O2/L

Nominal and measured concentrations:

Nominal loading rates (mg/L): 0.16, 0.40, 1.0, 2.5 and 6.4
Measured concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass vessels
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: fill volume 100 mL
- Aeration: N/a
- Type of flow-through (e.g. peristaltic or proportional diluter): N/a
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel:1 daphnid
- No. of vessels per concentration (replicates):5
- No. of vessels per control (replicates):5
- No. of vessels per vehicle control (replicates):
- Biomass loading rate:n/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days
- Light intensity: <500 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5 for main study
- Justification for using less concentrations than requested by guideline:
- Range finding study: Yes
- Test concentrations: 1.0. 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.98 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

The LOEL was 0.40 mg/L as the body length of the parental generation daphnids in this test group were significantly smaller (P<0.05) than the daphnids in the control group.
The NOEL was 0.16 mg/L as there were no significant mortalities (immobilization) observed in the parental generation (P1), there were no significant differences (P≥0.05) in terms of the number of live young produced per adult and the body length of the parental generation daphnids in this test group were not significantly different when compared to the control after 21 days.

Validity criteria fulfilled:

yes

Conclusions:

The LOEL was 0.40 mg/L as the body length of the parental generation daphnids in this test group were significantly smaller (P<0.05) than the daphnids in the control group.
The NOEL was 0.16 mg/L as there were no significant mortalities (immobilization) observed in the parental generation (P1), there were no significant differences (P≥0.05) in terms of the number of live young produced per adult and the body length of the parental generation daphnids in this test group were not significantly different when compared to the control after 21 days.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.98 mg/L