Reaction product of {Sulfonic acid derivatives of [4-(aminoalkylamino)-6-substituted-1,3,5-triazin-2-ylamino]benzene}, ammonia water, sodium chloride and [poly(1~4)chlorosulfonyl(poly(1~4) benzo-poly(3~0)pyrido-5,10,15,20-tetraazaporphyrinato- N21,N22,N23,N24)]copper(II)

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:

Experimental starting date: 06 November 2013; Experimental completion date: 25 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: CIM-25
Description: dark purple powder
Sponsors description: dark blue powder
Batch: MB-1
Expiry Date: 31 March 2014
Storage Conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
100 mg/L

- Sampling method:
Water samples were taken from the control and the 100 mg/L test group (replicates R1 – R4 pooled) at 0 and 48 hours for quantitative analysis.

- Sample storage conditions before analysis:
Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis ifnecessary.

Test solutions

Vehicle:

no

Details on test solutions:

RANGE-FINDING TEST:
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.

An amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 100 mg/L test concentration from which a series of dilutions was made to give the remaining test concentrations of 0.10, 1.0, and 10 mg/L.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 20°C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods.
Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized Daphnia magna were recorded.

The control group was maintained under identical conditions but not exposed to the test item.

A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

DEFINITIVE TEST:
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed.

EXPERIMENTAL PREPARATION:
An amount of test item (200 mg) was dissolved in test water and the volume adjusted to 2 liters to give the 100 mg/L test concentration.

The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

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. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable.

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at 20°C throughout the test.

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCI.
pH was recorded at the start and termination of the test.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of test.

Nominal and measured concentrations:

Definitive test:
Nominal concentration: 100 mg/l.
Measured concentration: 100% to 101% of nominal value.

Details on test conditions:

TEST SYSTEM/EXPOSURE CONDITIONS:
As in the range-finding test 250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared.

The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 20°C with a photoperiod of 16 hours light (628 to 673 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were not renewed during the exposure period.

TEST MEDIUM / WATER PARAMETERS
Reconstituted water (ISO medium) used for both the range-finding and definitive test is defined below:
Ingredient:
CaCl2.2H2O: 294 mg/L
MgSO4.7H2O: 123 mg/L
NaHCO3: 64.75 mg/L
KCl: 5.75 mg/L


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.

An estimate of the EC50 values was given by inspection of the immobilization data.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST:
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.

No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.

Chemical analysis of the 100 mg/L test preparation at 0 and 48 hours showed that measured concentrations of 104% and 103% of nominal were obtained respectively indicating that the test item was stable under test conditions.

Based on this information, a single test concentration of four replicates, of 100 mg/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed.

DEFINITIVE TEST:
Verification of Test Concentrations:
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 100% to 101% of nominal value and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentration only.

Immobilization Data:
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.

There was no immobilization in 20 daphnids exposed to a test concentration of 100 mg/L for a period of 48 hours. Inspection of the immobilization data gave the following results:
24 h EC50: >100 mg/L
48 h EC50: >100 mg/L

The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

VALIDATION CRITERIA:
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

WATER QUALITY CRITERIA:
The results of the water quality measurements are given in Table 3. Temperature was maintained at 20°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

OBSERVATIONS ON TEST ITEM SOLUBILITY:
Throughout the duration of the test, the test preparation was observed to be a dark blue solution.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the immobilization data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 24 and 48 hours based on the nominal test concentrations gave the following results:

24 hours:
EC50: 1.0 mg/L (95% CL 0.91 - 1.2 mg/L)
NOEC: 0.56 mg/L
LOEC: 1.0 mg/L

48 hours:
EC50: 0.71 mg/L (95% CL 0.65 - 0.76 mg/L)
NOEC: 0.32 mg/L
LOEC: 0.56 mg/L

The No Observed Effect Concentration is based upon zero immobilization at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Any other information on results incl. tables

Table 1: Cumulative Immobilization Data in the Range-finding Test:

Nominal Concentration	Cumulative Immobilized Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.10	0	0
1.0	0	0
10	0	0
100	0	0

Table 2: Cumulative Immobilization Data in the Definitive Test:

Nominal Concentration (mg/L)	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)
	24 Hours			48 Hours
	No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control R1	0	0	0	0	0	0
Control R2	0			0
Control R3	0			0
Control R4	0			0
100 R1	0	0	0	0	0	0
100 R2	0			0
100 R3	0			0
100 R4	0			0

 

Table 3: Water Quality Measurements:

Nominal Concentration (mg/L)	0 Hours			24 Hours	48 Hours
	pH	Mg O2/L	T°C	T°C	pH	Mg O2/L	T°C
Control (R1)	8.1	9.3	20	20	7.9	9.0	20
100 (R1)	8.1	9.2	20	20	8.0	8.9	20

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

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 the 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:

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100 mg/L for 48 hours at a temperature of 20°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results:

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 100% to 101% of nominal value and so the results are based on nominal test concentrations only.

Exposure of Daphnia magna to the test item gave an EC₅₀ value of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.