Benzenamine, 4-[(4-aminophenyl)(4-imino-2,5-cyclohexadien-1-ylidene)methyl]-, N-Me derivatives, molybdatephosphates

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:

08 January 2018 to 19 April 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: Batch no 982432, supplied by Client
- Expiration date of the lot/batch: 01 July 2018
- Purity test date: not stated

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: Not stated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: WAF's prepared
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentration and stability of the test item in the test preparation was verified by chemical analysis on Days 0, 7, 14 and 18 (fresh media) and on Days 2, 9, 16 and 21 (old media)
- Sampling method: Not stated
- Sample storage conditions before analysis: Samples were stored frozen prior to analysis. Duplicate sets of samples were taken on each occasion and stored frozen for further analysis if necessary.

Vehicle:

yes

Remarks:

Elendt M7 medium

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
-Nominal amounts of test item (20, 20 and 200 mg) were separately added to the surface of 20, 2 and 2 liters of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixtures allowed to stand for 1 hour. As a precautionary measure, it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75 to 100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. Observations on the media conducted after filtration suggested that no undissolved material remained in solution.
In

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: N/a
- Justification for species other than prescribed by test guideline:
- Source: In house laboratory cultures
- Age of parental stock (mean and range, SD): Ist instar
- Feeding during test yes
- Food type: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
- Amount: not stated
- Frequency: Daily

ACCLIMATION
- Acclimation period: N/a
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): N/a


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

N/a

Hardness:

244 TO 280 mg/L CaCO3

Test temperature:

19 °C to 22 °C

pH:

7.7 to 8.6

Dissolved oxygen:

8.8 tp 10.0 mg O2/L

Salinity:

n/A

Nominal and measured concentrations:

1.0, 1.8, 3.2 and 5.6 mg/L nominal loading rates
All measured test concentrations below the LOQ (0.23 mg/L) on all occassions except on Day 0 at 5.6 mg/L WAF when 0.29 mg/L was determined for both fresh and aged solutions

Details on test conditions:

TEST SYSTEM
- Test vessel:150 mL glass vessel
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: fill volume 100 mL
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/a
- Renewal rate of test solution (frequency/flow rate): Test solution renewed three times per week throughout the test
- No. of organisms per vessel: one
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates):0
- Biomass loading rate: N/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: All details given in Annex 2 of the report
- 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:The pH of the prepared media was 7.9 ±0.3
- 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: <1500 LUX

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: APPROX 1.8
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 1,10, 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

0.56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOELR

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.:

0.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

0.93 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.55 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

0.88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

For the parental Generation (P1):
Mortality (immobilization) occurred predominantly at the highest test loading rates of 1.8, 3.2 and 5.6 mg/L resulting in 100% mortality by Days 13, 2 and 2 respectively. Significant mortality (immobilization) also occurred throughout the test in the 1.0 mg/L loading rate WAF group resulting in 50% mortality by Day 15.
Mortality was also observed at the test loading rate of 0.56 mg/L on Day 4. However, statistical analysis of the mortality data using the Step-down Cochran-Armitage Test Procedure showed that the observed mortality in the 0.56 mg/L loading rate WAF group was not significantly different (P≥0.05) when compared to the control group, consequently, this mortality was classed as an inadvertent mortality for the purposes of the statistical analysis.
Some adult daphnia in the 0.56 mg/L loading rate were observed to be small and pale on Day 15, 19 and 20, in the 1.0 mg/L loading rate test group on Days 12, 13 and 15 to 20, and in the 1.8 mg/L loading rate test group on Day 12.
The daphnids at the remaining test concentrations were observed to be in the same general condition as the control daphnids.
The results for the length of the surci==viving adults showed that there were statistically significant differences (P≥0.05) between the control and the 0.56, 1.0, 1.8, 3.2 and 5.6 mg/L test groups in terms of length of the daphnids after 21 days exposure to the test item.

For effects on reproduction - after 21 days there were no statistically significant differences between the control and the 0.56 mg/L loading rate WAF group in terms of the number of live young produced per adult. The 1.0, 1.8, 3.2 and 5.6 mg/L loading rate WAF groups showed a statistically significant difference from the control after 21 days in terms of producing fewer numbers of live young per adult.

Effects on the Filial Generation (F1)
an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test with the exception of young produced by one replicate in the 0.56 mg/L loading rate on Day 12 where small young were observed, and on Day 15 when pale young were observed, in the 1.0 mg/L loading rate group on Day 12 where small and pale young were observed and in the 1.8 mg/L loading rate on Day 12 were small and pale young were observed.
Young were first produced in the control test group on Day 8 of the test.
Due to the toxic effect of the test item the parental generation (P1) of the 3.2 and 5.6 mg/L loading rate WAF groups were eliminated prior to the production of young.
There were no unhatched eggs or dead young in any control or treatment groups surviving to maturation.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item resulted in significant mortalities at the loading rates of 1.0, 1.8, 3.2 and 5.6 mg/L resulting in 50%, 100%, 100% and 100% mortalities by Days 15, 13, 2 and 2 respectively.
The 21-Day EL50 (immobilization) value, based on nominal loading rates, for the parental daphnia generation (P1) was calculated to be 0.93 mg/L with 95% confidence limits of 0.72 – 1.2 mg/L.
The 21-Day EL50 (reproduction) based on nominal loading rates was 0.88 mg/L with 95% confidence limits of 0.80 – 0.97 mg/L.
The Lowest Observed Effect Loading Rate and the No Observed Effect Loading Rate based on nominal loading rates were 1.0 and 0.56 mg/L respectively.

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

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 Source Substance Target Substance
Chemical name Benzenamine, N,N-dimethyl-, oxidized, molybdatetungstatephosphates Benzenamine, 4-[(4-aminophenyl)(4-imino-2,5-cyclohexadien-1-ylidene)methyl]-, N-Me derivatives, molybdatephosphates
IUPAC name reaction product of:
(a) [4-[bis[4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]-dimethyl-ammonium
(b) [4-[[4-(dimethylamino)phenyl]-[4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]-dimethyl-ammonium
(c) [4-[bis[4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]-dimethyl-ammonium
(d) phosphomolybdic acid
CAS No 101357-19-1 67989-22-4
EC No 309-916-8 268-006-8
Molecular weight range ≥ 2200 ≤ 3300 3800 - 7300
Molecular formula Reaction mass of C25 H30 N3. P Moy Wz O40 (y + z = 12), C24 H28 N3. P Moy Reaction mass of (C25H30N3)x.O3.P2O5.(MoO3)y; (C24H28N3)x.O3.P2O5.(MoO3)y and (C23H26N3)x.O3.P2O5.(MoO3)y
Wz O40 (y + z = 12) and C23 H26 N3. P Moy Wz O40 (y + z = 12) x = 6-10
y = 10-24
Structural formula

3. ANALOGUE APPROACH JUSTIFICATION
The target substance CAS 67989-22-4 is an organometallic multi-constituent substance. The source substance CAS 101357-19-1 has the same organic constituent (see table above) as the target substance consisting of triphenylmethan and three dimethylamine moieties. The inorganic Part B is very similar between source and target substances, consisting of the elements O, Mo, P and W in different compositions. 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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch no 982432, supplied by Client
- Expiration date of the lot/batch: 01 July 2018
- Purity test date: not stated

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: Not stated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: WAF's prepared
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentration and stability of the test item in the test preparation was verified by chemical analysis on Days 0, 7, 14 and 18 (fresh media) and on Days 2, 9, 16 and 21 (old media)
- Sampling method: Not stated
- Sample storage conditions before analysis: Samples were stored frozen prior to analysis. Duplicate sets of samples were taken on each occasion and stored frozen for further analysis if necessary.

Vehicle:

yes

Remarks:

Elendt M7 medium

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
-Nominal amounts of test item (20, 20 and 200 mg) were separately added to the surface of 20, 2 and 2 liters of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 95 hours and the mixtures allowed to stand for 1 hour. As a precautionary measure, it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75 to 100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. Observations on the media conducted after filtration suggested that no undissolved material remained in solution.
In

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: N/a
- Justification for species other than prescribed by test guideline:
- Source: In house laboratory cultures
- Age of parental stock (mean and range, SD): Ist instar
- Feeding during test yes
- Food type: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
- Amount: not stated
- Frequency: Daily

ACCLIMATION
- Acclimation period: N/a
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): N/a


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

N/a

Hardness:

244 to 280 mg/L CaCO3

Test temperature:

19 °C to 22 °C

pH:

7.7 to 8.6

Dissolved oxygen:

8.8 - 10.0 mg O2/L

Salinity:

n/A

Nominal and measured concentrations:

1.0, 1.8, 3.2 and 5.6 mg/L nominal loading rates
All measured test concentrations below the LOQ (0.23 mg/L) on all occassions except on Day 0 at 5.6 mg/L WAF when 0.29 mg/L was determined for both fresh and aged solutions

Details on test conditions:

TEST SYSTEM
- Test vessel:150 mL glass vessel
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: fill volume 100 mL
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/a
- Renewal rate of test solution (frequency/flow rate): Test solution renewed three times per week throughout the test
- No. of organisms per vessel: one
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates):0
- Biomass loading rate: N/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: All details given in Annex 2 of the report
- 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:The pH of the prepared media was 7.9 ±0.3
- 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: <1500 LUX

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: APPROX 1.8
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 1,10, 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

0.56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOELR

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.:

0.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

0.93 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.55 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

0.88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

For the parental Generation (P1):
Mortality (immobilization) occurred predominantly at the highest test loading rates of 1.8, 3.2 and 5.6 mg/L resulting in 100% mortality by Days 13, 2 and 2 respectively. Significant mortality (immobilization) also occurred throughout the test in the 1.0 mg/L loading rate WAF group resulting in 50% mortality by Day 15.
Mortality was also observed at the test loading rate of 0.56 mg/L on Day 4. However, statistical analysis of the mortality data using the Step-down Cochran-Armitage Test Procedure showed that the observed mortality in the 0.56 mg/L loading rate WAF group was not significantly different (P≥0.05) when compared to the control group, consequently, this mortality was classed as an inadvertent mortality for the purposes of the statistical analysis.
Some adult daphnia in the 0.56 mg/L loading rate were observed to be small and pale on Day 15, 19 and 20, in the 1.0 mg/L loading rate test group on Days 12, 13 and 15 to 20, and in the 1.8 mg/L loading rate test group on Day 12.
The daphnids at the remaining test concentrations were observed to be in the same general condition as the control daphnids.
The results for the length of the surci==viving adults showed that there were statistically significant differences (P≥0.05) between the control and the 0.56, 1.0, 1.8, 3.2 and 5.6 mg/L test groups in terms of length of the daphnids after 21 days exposure to the test item.

For effects on reproduction - after 21 days there were no statistically significant differences between the control and the 0.56 mg/L loading rate WAF group in terms of the number of live young produced per adult. The 1.0, 1.8, 3.2 and 5.6 mg/L loading rate WAF groups showed a statistically significant difference from the control after 21 days in terms of producing fewer numbers of live young per adult.

Effects on the Filial Generation (F1)
an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test with the exception of young produced by one replicate in the 0.56 mg/L loading rate on Day 12 where small young were observed, and on Day 15 when pale young were observed, in the 1.0 mg/L loading rate group on Day 12 where small and pale young were observed and in the 1.8 mg/L loading rate on Day 12 were small and pale young were observed.
Young were first produced in the control test group on Day 8 of the test.
Due to the toxic effect of the test item the parental generation (P1) of the 3.2 and 5.6 mg/L loading rate WAF groups were eliminated prior to the production of young.
There were no unhatched eggs or dead young in any control or treatment groups surviving to maturation.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item resulted in significant mortalities at the loading rates of 1.0, 1.8, 3.2 and 5.6 mg/L resulting in 50%, 100%, 100% and 100% mortalities by Days 15, 13, 2 and 2 respectively.
The 21-Day EL50 (immobilization) value, based on nominal loading rates, for the parental daphnia generation (P1) was calculated to be 0.93 mg/L with 95% confidence limits of 0.72 – 1.2 mg/L.
The 21-Day EL50 (reproduction) based on nominal loading rates was 0.88 mg/L with 95% confidence limits of 0.80 – 0.97 mg/L.
The Lowest Observed Effect Loading Rate and the No Observed Effect Loading Rate based on nominal loading rates were 1.0 and 0.56 mg/L respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.56 mg/L