[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2012-08-21 to 2012-31-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

(since the test item is poorly water soluble, the standard method for the preparation of aqueous media was modified. The test was performed with a saturated solution of the test item)

Qualifier:

according to guideline

Guideline:

EPA OPP 72-2 (Aquatic Invertebrate Acute Toxicity Test)

Deviations:

yes

Remarks:

(since the test item is poorly water soluble, the standard method for the preparation of aqueous media was modified. The test was performed with a saturated solution of the test item)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

PHYSICO-CHEMICAL PROPERTIES
- Water solubility (under test conditions): negligible

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 1, 3.2, 10, 32 and 100 % v/v (nominales)
- Sampling method: at 0 and 48 hours for quantitative analysis and pooled.
- Sample storage conditions before analysis: approximately -20°C prior to analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method of preparation: Saturation of the experimental solution:
550 mg of test item was dispersed in 11 liters of reconstituted water (Elendt M7 medium)
Stirring with a propeller at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped
Filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to remove undissolved test item
Obtention of a 100% v/v saturated solution.
Series of dilution to give further test concentration of 32, 10, 3.2 and 0.01 %v/v saturated solution.
Concentrations of the 2 ions of the test item were verified by the chemical analysis at 0 and 48 hrs.

- Application: daphnies were placed in glass jars containing 200 mL of the saturated solution test

- Controls: control group maintained under identical conditions but not exposed to the test item
- Vehicle: none
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory cultures
- Age at study initiation: less than 24 hours old
- Method of breeding:
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. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. 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. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome ofthe study.
- Feeding during test: no


ACCLIMATION
- Acclimation period: no (in house laboratory cultures)

QUARANTINE (wild caught)
- Duration:no quarantine

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

250 mg/L as CaCO3

Test temperature:

20°C to 21°C

pH:

See table 1: pH and dissolved oxygen measurements

Dissolved oxygen:

See table 1: pH and dissolved oxygen measurements

Nominal and measured concentrations:

Nominal test concentration, in % v/v saturated solution :
a - control
b- 1.0 % v/v
c- 3.2 % v/v
d- 10 % v/v
e- 32 % v/v
f- 100 % v/v

Corresponding Measured test concentrations (Geometric mean between 0-hour and 48-hour measured test concentration) :
a - control
b- 0.0051 mg/L
c- 0.019 mg/L
d- 0.058 mg/L
e- 0.16 mg/L
f- 0.66 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: jar
- Type : closed
- Material, size, headspace, fill volume: 250 mL glass jars
- Aeration: no
- Renewal rate of test solution: no renewal
- No. of organisms per vessel: 5 organisms
- No. of vessels per concentration (replicates): 4 replicates
- No. of vessels per control (replicates): 4 replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water Elend M7 medium (see table 2 for composition).

OTHER TEST CONDITIONS
- Adjustment of pH: yes, for dilution water
- Photoperiod: 16 hours light, 8 hour darkness with 20 minutes dawn and dusk transition period
- Light intensity: 479 to 513 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ~3
- Range finding study: yes
- Test concentrations: 0.1, 1 and 100 % v/v saturated solution
- Results used to determine the conditions for the definitive study: cumulative immobilization data from the range-finding study

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr207)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.011 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (0.0094 - 0.013 mg/l)

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.005 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

ca. 0.019 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Observation on test item solubility:
The control, 1.0 and 3.2% v/v saturated solution test preparations were observed to be clear colorless solutions for the duration of the test whilst the 10, 32 and 100 % v/v saturated solution test preparations were observed to be very slightly purple solutions, slightly purple solutions and purple solutions respectively for the duration ofthe test.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 - 24 hr = 0.75 mg/l (IC 95% 0.56 - 1 mg/l)
- EC50 - 48 hr = 0.45 mg/l (IC 95% 0.42 - 0.48 mg/l)

Reported statistics and error estimates:

It was not possible to calculate the slope and error of response curve at 24 and 48 hr.

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions:
* 48h - EC50 = 0.011 mg/L
* 48h - NOEC = 0.0051 mg/L
* 48h - LOEC = 0.019 mg/L
(geometric mean of measured test concentration)

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna.The method followed was designed to be compatible with OECD Guidelines. for Testing of Chemicals (April 2004) No 202,"Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

 

Methods

Information provided by the Sponsor indicated the water solubility of the test item to be negligible. Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

 

The test item is a precipitated reaction product of constituent 1 (cons. 1) and constituent 2 (cons. 2). Assuming that the test item was a 1:1 stoichiometric mixture of the two components, based on the percentage content of each component, equivalent test item concentrations could be determined.

 A pre-study media preparation trial indicated that the use of a saturated solution method of preparation followed by the removal of any undissolved test item by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) was appropriate for this test item.

 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution for 48 hours at a temperature of 20°C to 21°C under static test conditions.

The test item solutions were prepared by stirring an excess (50 mg/L) of test item in test medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Gelman Acrocap filter, first approximate 500 mL discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. The number of immobilized Daphnia were recorded after 24 and 48 hours.

 

Results

Analysis of the test preparations at 0 hours showed measured cons. 1 concentrations to range from 0.0054 to 0.49 mg/L (equivalent to test item concentrations of 0.011 to 1.0 mg/L). At 48 hours measured cons. 1 concentrations in the range of 0.0054 to 0.38 mg/L (equivalent to test item concentrations of 0.011 to 0.80 mg/L) were obtained.

Analysis of the test preparations at 0 hours showed measured cons. 2 concentrations to range from 0.0027 to 0.40 mg/L (equivalent to test item concentrations of 0.0051 to 0.77 mg/L). A slight decline in measured cons. 2 concentrations was observed at 48 hours in the range of 0.0027 to 0.29 mg/L (equivalent to test item concentrations of 0.0051 to 0.56 mg/L).

Given that variability in results obtained for both components, it was considered appropriate to base the results on the equivalent test item concentrations from analysis of cons. 2 only in order to give a worst case analysis of the data.

 

Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentrations:

-         EC50 – 48 hr = 0.011 mg/L (IC_95%0.0094 – 0.013 mg/L)

-         NOEC = 0.0051 mg/l

-         LOEC = 0.019 mg/l

Description of key information

LC50(48h) for freshwater invertebrates (Daphnia magna): 0.011 mg/L (OECD TG 202; static).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.011 mg/L

Additional information

The EC50 (48 hours) was 0.011 mg/L (measured concentrations) with 95% confidence limits of 0.0094 -0.013 mg/L.

The NOEC was 0.0051 mg/L