(p-ammoniophenyl)bis(2-hydroxyethyl)ammonium sulphate

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 Apr. 18, 2013 to Apr. 20, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to OECD principles of GLP

Specific details on test material used for the study:

The test substance was Colipa A050 (CAS #54381-16-7; Batch Number 20110103), also known as G11-A11117 (Blauentwickler); 4-(Di(2-hydroxyethyl)amino)-Anilin-sulfat (1:1), hydrat; 2,2`-[4-aminophyenyl))imino]bis(ethanol) sulphate; Ethanol, 2,2`-[(4-aminophenyl))imino]bis-,sulphate (1:1) (salt); 2-[[(4-aminophenyl)-(2-hydroxyethyl)]amino]ethanol sulphate; INCI name: N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate.
The Certificate of Analysis reported the active ingredient in the form of free base for Colipa A050 as 62.5% by weight. Consequently the data in this record are expressed as active ingredient in conformity with the study report.
Since the active ingredient is not the registered substance a new Certificate of Analysis was performed of the tested substance, N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulphate monohydrate, with a purity level of 99.24%. So the final results of the study were re-calculated taking into account the revised purity level and the EC50 is expressed as the tested substance.”

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 0.15, 0.33, 0.73, 1.6, 3.6, and 8.0 mg a.i./L.
- Sampling method: The concentration of Colipa A050 was measured in the control and all test substance treatments in samples collected from parent solutions at initiation and 24 hours, and in samples composited from spent replicate solutions at 24 and 48 hours. At each sampling point, 9 mL samples were collected into appropriately labeled culture tubes. The samples were diluted to 10 mL with 2% erythorbic acid and further diluted, if necessary, with 0.2% erythorbic acid to provide final concentrations within the analytical standard concentration range (i.e., 0.0150 to 0.600 mg a.i./L). Two quality control (QC) fortification spikes at concentrations that bracket the expected high and low test substance treatment concentrations were prepared and analyzed in a similar manner.
- Sample storage conditions before analysis: The samples were placed in vials appropriate for use in HPLC-UV and were analyzed by HPLC-UV.

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTION
- Method: A primary standard solution of 0.0080 mg a.i./mL was prepared at test initiation by diluting approximately 0.0256 grams of Colipa A050 (0.0160 grams as active ingredient) to a volume of 2 L with dilution water. This primary standard solution was used as the highest test concentration and its appropriate aliquots were diluted to 1 L to prepare the test concentrations of 0.15, 0.33, 0.73, 1.6, and 3.6 mg a.i./L.
- Controls: The control consisted of dilution water only
- Evidence of undissolved material: The test substance was completely dissolved at all concentrations tested.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: Not reported
- Source: In-house daphnia culture maintained by ABC Laboratories, Inc
- Age of parental stock: < 24 h
- Feeding during test: No
- Culturing of daphnia: All daphnids were cultured in a temperature-controlled area at approximately 20˚C. During the culture period, the daphnids were fed a suspension of the algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) at least once a day supplemented by an artificial diet consisting of a wheat grass, salmon starter, and yeast suspension. Neonates for the definitive test were collected from a single culture containing adults that were approximately 13 days old.

ACCLIMATION
- Acclimation period: Since the culturing and testing environmental parameters were equivalent (i.e., temperature, dilution water, and lighting), no acclimation period was necessary.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

154 mg CaCO3/L

Test temperature:

19.3 to 19.9°C

pH:

8.2 to 8.5

Dissolved oxygen:

New Solutions: 8.4 to 9.0 mg/L (97 to 103% saturation)
Old Solutions: 7.2 to 8.8 mg/L (83 to 101% saturation)

Nominal and measured concentrations:

Nominal test concentrations: 0 (control), 0.15, 0.33, 0.73, 1.6, 3.6, and 8.0 mg a.i./L
Mean measured test concentrations: Mean measured test concentrations were 18 to 72% of nominal, and results are based on mean measured concentrations.
Details on geometric mean measured concentrations are provided in Table 1 under ‘Any other information on materials and methods incl. tables section’

Details on test conditions:

TEST SYSTEM
- Test vessel: Jars
- Type: Close (covered with PTFE lined screw caps)
- Material: 125 mL glass jars each containing approximately 125 mL of control or test substance treatment solution
- Aeration: No
- Renewal rate of test solution: The control and each treatment solutions were renewed after 24 hours of exposure.
- No. of organisms per vessel: 5 per vessel
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- Biomass loading rate: 1 daphnid/ 25 mL test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was moderately hard freshwater prepared by blending naturally hard well water with well water that was demineralized by reverse osmosis. These waters were blended to yield a total hardness of approximately 130 to 160 mg CaCO3/L. Prior to use; the dilution water was then passed through a sediment filter. The dilution water for the range-finding test was also UV irradiated and aerated prior to use. Characterization of the base water, i.e. ABC well water, used to prepare the dilution water is provided in the study report.
- Total organic carbon of the dilution water: < 2.0 mg/L
- Alkalinity (at test initiation): 168 mg CaCO3/L
- Conductivity (at test initiation): 368 µS
- Intervals of water quality measurement: The water quality parameters (i.e. total hardness, total alkalinity, and conductivity) were measured in the dilution water at test initiation. The temperature, dissolved oxygen concentration, and pH were measured daily in all replicates.

OTHER TEST CONDITIONS
- Adjustment of pH: Not reported
- Photoperiod: 16 hours fluorescent light and 8 hours dark with two 30-minute simulated dawn and dusk periods.
- Light intensity: 539 lux

EFFECT PARAMETERS MEASURED: The daphnids were observed for immobility and sublethal effects at approximately 24 and 48 hours after test initiation.
- RANGE FINDING STUDY: Yes
- Nominal test concentrations: 0, 0.10, 1.0, 10, and 100 mg a.i./L
- Results used to determine the conditions for the definitive study: Yes, at test termination, after 48 hours of exposure, immobility was 0, 0, 40, 100, and 100% in the 0 (control), 0.10, 1.0, 10, and 100 mg a.i./L test substance treatments, respectively. All daphnids appeared normal with no behavioral or morphological abnormalities.
- Evidence of any colored tinge in solution: The control and test substance treatment solutions < 10 mg a.i./L were clear and colorless throughout the test. The 10 mg a.i./L test solution was clear and colorless at initiation and clear with a pink tint at 24 hours. The 100 mg a.i./L test solution was clear with a purple tint at initiation and clear with a brown tint at 24 hours The control solutions were clear and colorless throughout the test with no visible particulates, surface films, precipitates, or undissolved test substance.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.381 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Confidence Interval 0.346 - 0.419 mg/L

Details on results:

-The EC50 after 24h and 48h initially calculated as 0.675 and 0.24 mg a.i./L, were recalculated as 1.1 and 0.381 mg/l respectively, based on the tested substance and taking into account the updated certificate of analysis (see the explanations provided in "Specific details on test material used for the study").
- Mortality in test treatment group: % Immobility observed at 0.0374, 0.0687, 0.133, 0.477, 1.47, and 5.76 mg a.i./L was 0, 0, 5, 100, 100, and 100% respectively at 48 hours exposure period.
-Mortality in control: 0%
-Sublethal effects: No sublethal effects were noted during 48 hours exposure period.
Further details on immobility is given in Table 1 “Any other information on results incl. tables”

Reported statistics and error estimates:

Fisher’s exact test (p < 0.05) was used to calculate statistically significant difference in immobility as compared to the control.

Table 1: Immobility of Daphnia magna exposed to Colipa A050 for 48 Hours under static-renewal test conditions (Study # 68339)

Geometric Mean Concentration (mg a.i./L)	Treatment Mean Percent Immobile (48 Hours)
0 (Control)	0
0.0374	0
0.0687	0
0.133	5
0.477	100*
1.47	100*
5.76	100*

* Statistically significant difference in immobility as compared to the control (Fisher’s Exact Test,p< 0.05)

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour EC50 of N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate to Daphnia magna was 0.381 mg/L (confidence interval 0.346 - 0.419 mg/L), based on geometric mean measured concentrations.

Executive summary:

The 48 hour EC50 of N,N-Bis(2 -hydroxyethyl)-p-phenylenediamine sulfate to Daphnia magna was determined under semi-static conditions following procedures as described in OECD guideline 202 (acute toxicity for daphnia) and according to GLP.

The nominal test concentrations were 0 (control), 0.15, 0.33, 0.73, 1.6, 3.6, and 8.0 mg active ingredient/L (the active ingredient being the tested substance without sulphate and monohydrate). The geometric mean measured test concentrations were <MQL (Control), 0.0374, 0.0687, 0.133, 0.477, 1.47, and 5.76 mg a.i./L (18 to 72% of nominal). There were 4 replicates per test concentration with 5 daphnia/ replicate.

The effect parameters observed were immobility and sublethal responses. The results were based on geometric mean measured concentration.

% Immobility observed at 0, 0.0374, 0.0687, 0.133, 0.477, 1.47, and 5.76 mg a.i./L was 0, 0, 0, 5, 100, 100, and 100% respectively at 48 hours exposure period. All the validity criteria were met.

The 48 hour EC50 of N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate to Daphnia magna was 0.381 mg/L based on geometric mean measured concentrations of the tested substance (equivalent to 0.240 mg a.i./L), with a confidence interval of 0.346 - 0.419 mg/L.

This acute toxicity test is classified as acceptable and satisfies the guideline requirement of OECD 202 acute toxicity for daphnia.

Description of key information

The 48 hour EC50 of N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate to Daphnia magna was 0.381 mg/L (confidence interval 0.346 - 0.419 mg/L), based on geometric mean measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.381 mg/L

Additional information

The 48 hour EC50 of N,N-Bis(2 -hydroxyethyl)-p-phenylenediamine sulfate toDaphnia magnawas determined under semi-static conditions following procedures as described in OECD guideline 202 (acute toxicity for daphnia) and according to GLP.

The nominal test concentrations were 0 (control), 0.15, 0.33, 0.73, 1.6, 3.6, and 8.0 mg active ingredient/L (the active ingredient being the tested substance without sulphate and monohydrate). The geometric mean measured test concentrations were <MQL (Control), 0.0374, 0.0687, 0.133, 0.477, 1.47, and 5.76 mg a.i./L (18 to 72% of nominal). There were 4 replicates per test concentration with 5 daphnia/ replicate.

The effect parameters observed were immobility and sublethal responses. The results were based on geometric mean measured concentration.

% Immobility observed at 0, 0.0374, 0.0687, 0.133, 0.477, 1.47, and 5.76 mg a.i./L was 0, 0, 0, 5, 100, 100, and 100% respectively at 48 hours exposure period.All the validity criteria were met.

The 48 hour EC50 of N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate toDaphnia magnawas 0.381 mg/L based ongeometric mean measured concentrations of the tested substance (equivalent to 0.240 mg a.i./L),with aconfidence interval of 0.346 - 0.419 mg/L.